Development of a Lateral Flow Strip with a Positive Readout for the On-Site Detection of Aflatoxin B1

Aflatoxin B1 is one of the contamination indicators for food safety monitoring. The rapid and effective assessment and determination of AFB1 in food is of great importance to dietary safety. The lateral flow assay shows advantages in its simplicity, and rapidity, and provides a visual readout, while the available lateral flow assay for AFB1 requires a competitive format that produces readings inversely proportional to the AFB1 concentration, which is counterintuitive and may lead to a potential misinterpretation of the results. Herein, we developed a positive readout aptamer-based lateral flow strip (Apt-strip) for the detection of AFB1. This Apt-strip relies on the competition between AFB1 and fluorescein-labeled complementary DNA strands (FAM-cDNA) for affinity binding to limited aptamers against AFB1 (AFB1-Apt). In the absence of AFB1, AFB1-Apt hybridizes with FAM-cDNA. No signal at the T-line of the Apt-strip was observed. In contrast, AFB1-Apt binds to AFB1 in the sample, and then a part of the FAM-cDNA is hybridized with the free AFB1-Apt, at which time the other unreacted FAM-cDNA is captured by A35-Apt on the T-line. The signal was observed. This method achieved fast detection of AFB1 with a detection limit (DL) of 0.1 ng/mL, positive readout, and increased sensitivity.


Introduction
Aflatoxin B 1 (AFB 1 ), a fungal metabolite, is highly toxic and carcinogenic to humans and animals [1,2], and it is most commonly found in cereal and oil foods [3]. Long-term exposure to very low levels of AFB 1 in feed and food is a threat to human and animal health [4,5]. In order to protect the health of humans and animals, many countries and regions have set the maximum allowable limit of AFB 1 (Table S1) in feed and food [6,7]. The National Food Safety Standards of China (GB 2761-2017) stipulate that AFB 1 is one of the compulsory inspection items for most foods. Therefore, it is particularly important to establish an accurate and rapid method for the determination of AFB 1 in food. Currently, liquid-chromatography-based methods, including high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), are already officially accepted for the quantitative analysis of AFB 1 . However, these methods have the disadvantages of being time consuming, requiring expensive equipment and professional technicians to operate, and are not suitable for the rapid on-site screening of bulk samples. The enzyme-linked immunosorbent assay (ELISA) [8] is an alternative method for the rapid analysis of AFB 1 . Nevertheless, the antibodies are costly and not easily stored, which limit its application in the rapid analysis of AFB 1 .
Lateral flow assay (LFA) has been widely used for the rapid determination of AFB 1 [9][10][11][12] because of its simplicity, portability, cost effectiveness, and suitability for on-site screening [13][14][15][16]. In 2005, Delmulle et al. prepared an LFA strip using colloidal gold as a signal marker for the rapid assay of AFB 1 [17]. Subsequently, there has been a significant increase in the number of studies based on colloidal gold LFA for the detection of AFB 1 [18][19][20]. Until 2014, Wang first reported the use of luminescent nanomaterials as signal amplifiers for the more sensitive detection of AFB 1 [21]. Since then, various luminescent materials, including quantum dots [22][23][24][25] and fluorescent microspheres [26,27], have been used as signal probes for the LFA technique to enhance the sensitivity of AFB 1 detection. Recently, in view of the high cost and fallibility of antibodies, aptamer-based lateral flow strips have been developed to detect AFB 1 [28][29][30]. However, the currently reported LFA methods for detecting AFB 1 produce readings inversely proportional to the analyte content; i.e., negative samples have the strongest T-line signal intensity, while positive samples have a decreasing T-line signal intensity with an increasing AFB 1 concentration. This is counterintuitive [31]. In particular, when the AFB 1 concentration is at the critical value, the sensitivity is low and the observations are not intuitive.
In this paper, we proposed a positive readout aptamer-based lateral flow strip (Aptstrip) for the detection of AFB 1 . The Apt-strip indirectly assays AFB 1 utilizing the competition of AFB 1 and 6-FAM labeled DNA complementary strands (FAM-cDNA) to the affinity binding to AFB 1 -Apt. This Apt-strip presents a positive readout. With the increase in AFB 1 concentration, the greater the amount of FAM-cDNA hybridized at the T-line, and the stronger the fluorescence intensity showed. Through using this Apt-strip, AFB 1 can be rapidly detected within 15 min, and the detection limit (DL) is less than 0.1 ng/mL. The method possesses the characteristics of good selectivity, a strong anti-interference ability, high sensitivity, and the potential for the rapid and on-site screening of AFB 1 in the food matrix.

Principle of the Apt-Strip
The principle of the Apt-strip is shown in Scheme 1. FAM-cDNA is loaded on the conjugate pad, and the streptavidin-labeled aptamer against AFB 1 is immobilized at the T-line (Scheme 1a). For AFB 1 -positive samples analysis, AFB 1 competes with FAM-cDNA to bind to the affinity ligand AFB 1 -Apt in a solution, forming the AFB 1 -Apt/AFB 1 complex. Then, the AFB 1 -Apt/AFB 1 complex and free FAM-cDNA probe migrate to the NC membrane, and the A35-Apt of the T-line hybridized with the free FAM-cDNA probe, resulting in the formation of double-stranded DNA (hybridized A35-Apt/FAM-cDNA probe) and the immobilization of FAM-cDNA at the T-line. As a result, a fluorescent spot is observed at the T-line with a ChemiDocTM MP system (Scheme 1b). The fluorescence intensity is increased by increasing the concentration of AFB 1 , which can be used for the quantitative analysis of AFB 1 . For negative samples, the free FAM-cDNA probes hybridize with AFB 1 -Apt in a solution to form double-stranded DNA (AFB 1 -Apt/FAM-cDNA probe) instead of A35-Apt of T-line, leading to a negative fluorescence signal at the T-line.
The results of the qualitative analysis can be identified by the naked eye with the aid of the ChemiDocTM MP system. For quantitative results, this is achieved by further analysis of the image. The specific steps are as follows. First, the strip is imaged using the ChemiDocTM MP system. Then, the fluorescence intensities are converted to numerical values with the help of software Image J. The relationship between the AFB 1 concentration (X) and fluorescent intensity (Y) can be obtained by constructing a fitting curve.

Optimization of cDNA Length
The affinity of the cDNA and aptamer is a vital factor for the competition of cDNA and AFB 1 to bind to the aptamer, which differs depending on the length of the cDNA [32][33][34][35][36]. For this reason, we optimized the length of cDNA, ranging from 10 to 16 nucleotides. As shown in Figure 1, with the increase in cDNA length (n), both blank samples and AFB 1 samples (100 nM) showed an increasing fluorescence intensity, which resulted from stronger hybridized double-stranded DNA between the cDNA with a longer length and aptamer. The biggest fluorescence intensity change induced by AFB 1 was obtained when cDNA with 12 nucleotides (12-cDNA). The results indicate that cDNA with more than 12 nucleotides is not in favor of the competition of AFB 1 . Thus, 12-cDNA was selected as the complementary strand for the subsequent tests.

Analytical Performance of the Apt-Strip
To verify the feasibility of the Apt-strip for the detection of AFB 1 , different concentrations of AFB 1 (0, 0.1, 1, 5, 10, 30, 60, and 100 ng/mL) were analyzed using the Apt-strip. Buffer C containing10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10 mM MgCl 2 , and 10% methanol was used as the assay buffer.
As shown in Figure 2a, there was no fluorescence signal at the T-line for the blank sample, and the fluorescence signal appeared when the AFB 1 concentration was 0.1 ng/mL. Then, the fluorescence intensity gradually increased with the increase in AFB 1 concentration, and the fluorescence intensity reached the highest when the AFB 1 concentration was 100 ng/mL. The calibration curve ( Figure 2c) was constructed using fluorescence intensity (Y) against the concentrations of AFB 1 (X). The fitted equation was Y = 11,891 − 11,751 × exp (−0.05X) with a reliable correlation coefficient (R 2 = 0.9864), and the dynamic range of AFB 1 was from 0.1 ng/mL to 100 ng/mL. The DL for the qualitative evaluation was defined as the minimum concentration when displaying a very weak fluorescence intensity at the T-line, compared with a blank sample [37]. Therefore, the DL was less than 0.1 ng/mL, which was comparable to the value previously reported using other antibody or other aptamer-based LFA (Table 1). In addition, the DL was less than the minimum allowable limit of AFB 1 (Table S1) set by different countries and regions. Therefore, the Apt-strip could meet the various screening requirements of AFB 1 . It is noteworthy that compared with other strips for AFB 1 detection, the Apt-strip showed a positive readout, which means the fluorescence signal increased with the increase in AFB 1 concentration. This positive readout method is more convenient and sensitive for qualitative analysis, especially for the analysis of samples containing very small amounts of AFB 1 . To the best of our knowledge, no AFB 1 detection method has been reported by positive readout strips, and this strategy is the first report of positive readout test strips for AFB 1 detection.  To assess the precision and accuracy of the prepared Apt-strip, we chose spiked samples of corn and wheat as the food matrixes. All of the food samples were first analyzed by the HPLC-FLD method to ensure there was no contamination of AFB 1 .
First, we accurately weighed 5 g of the sample into a 50 mL PP tube. Then, we added the appropriate amount of different concentrations of the AFB 1 standard solution. The spiked samples were processed as in Section 3.6. The extracts were analyzed using the Apt-strip. Experimental results in Table 2 indicate that the recoveries were in the range of 50.0-97.0% for AFB 1 with relative standard deviations (RSDs) less than 36.7%, and were acceptable within the requirements of No. 401/2006 [7]. These results suggest that the Apt-strip developed in this study can be used for the quantitative and qualitative detection of AFB 1 in real samples. To verify the specificity of the Apt-strip for AFB 1 , we tested several mycotoxins, including OTA, AFG 1 , AFG 2 , ZAE, and the mixture of AFB 1 with the above mycotoxins together. AFB 1 and other mycotoxins were all tested at 50 ng/mL. The results are shown in Figure 3. The tested mycotoxins did not cause a significant increase in intensity (Figure 3b), while AFB 1 induced a clear fluorescence spot (Figure 3a). The mixture of these mycotoxins with AFB 1 presented a similar fluorescence spot as for the AFB 1 sample. The results indicate that the Apt-strip had a good selectivity for AFB 1 detection. To evaluate the stability of the Apt-strip, stability experiments over time were carried out. Apt-strips of the same batch were placed in foil pouches with a desiccant, and were stored at room temperature for 90 days. Then, the strips were used to detect different concentrations of AFB 1 (0, 0.1, 1, 5, 10, 30, 60, and 100 ng/mL), and the detection was performed once every 24 h. The Apt-strip assayed different concentrations of AFB 1 , and the RSDs were all less than 5.3% (Table 3), indicating that the repeatability of the Aptstrip remained consistent. Trend plots with a 24 h interval ( Figure 4) show that there was no change in trend over time. These results show that the performance of the Apt-strip remained stable after 90 days of storage at room temperature.

Detection of AFB 1 in Real Samples
To evaluate the practicability of the Apt-strip in real samples, 25 batches real samples were analyzed using the Apt-strip and HPLC-FLD. First, 25 batches of real samples, composed of 13 corn, 6 wheat, and 6 sorghum, were collected from Changzhi City, Shanxi Province, China. Table 4 shows that 11 out of 25 samples were found to have AFB 1 . The residual level of AFB 1 ranged from 2.4 ± 0.7 µg/kg to 75.3 ± 5.3 µg/kg. All the samples were confirmed by HPLC-FLD analysis. The results of the Apt-strip were not false-positive or false-negative. Figure 5 indicates that the two methods yielded consistent results with a good correlation (R 2 = 0.9938). The above results demonstrate that the Apt-strip is reliable and accurate for thepractical qualitative and quantitative detection of AFB 1 in real samples, and is a portable tool for the on-site detection of AFB 1 .

Aptamer and DNA Probes
The aptamer sequence was referenced from our previously published paper [44]. The complementary DNA (cDNA) of the aptamer was designed for the development of LFA. Aptamer and cDNA were ordered from Sangon Biotechnology Co., Ltd. (Shanghai, China), and the sequences are listed in Table 5. A35 had one biotin label at the 3' terminus and a TEG (triethylene glycol) linker. The cDNA each had one 6-FAM label at the 3' terminus. Table 5. Sequences of aptamer and cDNA.

Preparation of A35-Apt Coated Microplates
The A35-Apt was coated on the surface of 96-well black microplates by the following steps. Firstly, 100 µL of streptavidin (SA, 10 µg/mL) in 0.1 M Na 2 CO 3 solution (pH 9.6) was added into the wells of the microplates and they were incubated overnight at 4 • C. Then, after washing three times with 150 µL of buffer A (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20), the wells of the microplate were bound with 200 µL of buffer A containing 10 mg/mL BSA at 25 • C for 2 h under shaking. The wells were washed with 250 µL of buffer A. Next, 100 µL of buffer A containing 25 nM biotinylated A35-Apt was added to the SA coated wells, and the mixture was incubated for 1 h at 25 • C under shaking. Finally, the wells were washed three times with 200 µL of buffer B (10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , and 50 mM NaCl), and the A35-Apt coated microplate was ready for the analysis of targets.

Optimization Procedure of cDNA Length
The optimization was as follows. Firstly, 100 µL of buffer B containing 100 nM AFB 1 was added to each of the four wells of the A35-Apt coated microplates, and 100 µL of 20 nM n-cDNA (n = 10, 12, 14, and 16) was added sequentially. Secondly, the mixtures were incubated for 10 min at room temperature, and the wells were washed three times with buffer B. Thirdly, 100 µL of buffer B was added sequentially to the four wells, and the fluorescence intensity was measured using a multifunctional enzyme marker (Infinite M Plex, λ ex/em = 495/517 nm). Meanwhile, buffer B without AFB 1 (AFB 1 0 nM) was used as a control.

Manufacture of the Apt-Strip
The structure of the Apt-strip is shown in Scheme 1a. Firstly, an NC membrane was treated with a streptavidin labeled aptamer against AFB 1 (A35-Apt-SA, 30 µM, 0.5 µL/cm) for T-line, and a conjugate pad was treated with cDNA (2 µM, 5 µL/cm). Then, the treated NC membrane and conjugate pad were dried at 37 • C for 20 min. Secondly, the test strips were assembled according to Scheme 1a, and the joints overlapped by 2 mm. Thirdly, the strip was cut into 4 mm wide test strips and stored in a desiccator at room temperature until use. In our experiments, we omitted the preparation of the control line (C-line) because it is very simple and always effective in actual operation [43]. Note that the final Apt-strip design would include a control line.

Assay Procedure of AFB 1 in Food Samples
The food samples were prepared according to the procedure described in GB 5009.22-2016 of China. Briefly, solid samples of food were ground, weighed (5 g), and transferred into a 50 mL PP tube, and then extracted with 20 mL methanol/water (70:30, v/v) by homogenizing for 5 min, followed by centrifugation at 5000 r/min for 5 min at 4 • C. The supernatant was collected and diluted seven times with buffer B to obtain a sample solution.
The procedure for determining AFB 1 using Apt-strip is as follows. Forst, 2 µL of AFB 1 -Apt (2 µM) and 50 µL of the sample solution were mixed in a PP tube and incubated at 25 • C for 5 min, and then the mixture was placed on the Apt-strip. After 10 min, the results were observed by the ChemiDocTM MP system. Then, the fluorescence intensity at the T-line was scanned with Image J software to achieve a quantitative analysis of the assay results. All of the assays were repeated three times.

HPLC-FLD Confirmation
The reliability and practicability of the Apt-strip was further confirmed by HPLC-FLD analysis using the retention time and chromatographic peak area of AFB 1 as the basic parameters. The specific analytical conditions are listed in the supporting information (HPLC-FLD conditions).

Conclusions
In this paper, we report a positive readout Apt-strip for the detection of AFB 1 in food. We used competition between AFB 1 and FAM-cDNA to bind the limited AFB 1 -Apt and free FAM-cDNA hybridized to A35-Apt at the T-line, and detected AFB 1 indirectly by measuring the fluorescence intensity at the T-line. The DL of this method was 0.1 ng/mL for AFB 1 in buffer and 0.3 ng/g in food, with a dynamic range of 0.1-100 ng/mL in the buffer and an R 2 of 0.9864. The sensitivity and selectivity were very satisfactory. The validation results on the spiked samples and real samples show that the method is dependable. In addition, the Apt-strips are low-cost, and the detection process does not require specialized technicians and it can be used as a rapid scanning tool for food industries or regulatory laboratories. This work is expected to provide new insight into the detection of other food contaminants.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27154949/s1, Table S1: The maximum tolerable limit of AFB 1 in feed and food set by different countries; HPLC-FLD conditions.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.