A Novel Graphene Oxide-Based Aptasensor for Amplified Fluorescent Detection of Aflatoxin M1 in Milk Powder

In this paper, a rapid and sensitive fluorescent aptasensor for the detection of aflatoxin M1 (AFM1) in milk powder was developed. Graphene oxide (GO) was employed to quench the fluorescence of a carboxyfluorescein-labelled aptamer and protect the aptamer from nuclease cleavage. Upon the addition of AFM1, the formation of an AFM1/aptamer complex resulted in the aptamer detaching from the surface of GO, followed by the aptamer cleavage by DNase I and the release of the target AFM1 for a new cycle, which led to great signal amplification and high sensitivity. Under optimized conditions, the GO-based detection of the aptasensor exhibited a linear response to AFM1 levels in a dynamic range from 0.2 to 10 μg/kg, with a limit of detection (LOD) of 0.05 μg/kg. Moreover, the developed aptasensor showed a high specificity towards AFM1 without interference from other mycotoxins. In addition, the technique was successfully applied for the detection of AFM1 in infant milk powder samples. The aptasensor proposed here offers a promising technology for food safety monitoring and can be extended to various targets.


Introduction
Aflatoxin M 1 (AFM 1 ), one of the most toxic mycotoxins, was moved from group 2B to group 1 carcinogens by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) [1,2]. AFM 1 can be encountered in dairy products as a hydroxylate metabolite derived from feeding dairy cows aflatoxin B 1 -contaminated feeds [3][4][5]. Since dairy products are an important nutrient for humans, especially for infants, the presence of AFM 1 in dairy products is one of the most serious hazards for food safety [6]. To protect humans from this health threat, many regulatory agencies have defined maximum residue levels (MRLs) for AFM 1 in dairy products [7,8]. In Brazil, China, and USA, the maximum level of AFM 1 in milk has been fixed to 0.5 µg/kg [9,10]. The European Commission Regulation has set much more restrictive limits, i.e., 0.05 µg/kg in milk products for adults, and this level is lowered to 0.025 µg/kg for baby and infant products [11]. Considering the 2.2. Fluorescent Response of the Amplified Aptasensor for AFM 1 In this amplification strategy, the FAM-labelled AFM 1 aptamer was diluted to 200 nM in Tris buffer (10 mM Tris, 120 mM NaCl, 5 mM KCl, 20 mM CaCl 2 , pH 7.0), and 20 µg mL −1 of GO was added to the working solution for 15 min at room temperature to form the aptamer/GO complex and quench the fluorescence. Subsequently, solutions at different concentrations of AFM 1 and DNase I (200 U) were simultaneously added to the aptamer/GO solutions, and the mixtures were and incubated at room temperature for 1 h. Afterwards, the fluorescence intensities of the mixtures were recorded using an F-7000 fluorophotometer (Hitachi, Tokyo, Japan). The emission spectra were measured in the range of 510 to 630 nm with the excitation wavelength at 480 nm, and slit widths for both of the excitation and the emission were set at 10 nm.

Specificity Analysis
To evaluate the specificity of this aptasensor for AFM 1 over other mycotoxins, four different mycotoxins (AFB 1 , OTA, ZEA and α-ZOL) were measured at the same concentration of 4 ng mL −1 . The other experimental procedures were the same as AFM 1 determination, and the changes of fluorescence intensity for these mycotoxins were compared.

Method Validation
The feasibility and practicability of this sensing platform was verified by the quantitative detection of AFM 1 in infant milk powder samples. The samples were spiked with AFM 1 at 0, 1.5, 2.5 and 5 µg/kg (three replicates per treatment). Each sample was accurately weighed (0.5 g) into 10 mL centrifuge tubes. Then, 2.5 mL of extraction solution (70% methanol in water) was added to extract AFM 1 from the samples. The entire mixture was vortexed using Vortex-Genie 2 (Scientific Industries, Bohemia, NY, USA) for 5 min and then centrifuged at 10,000 g for 10 min. The supernatant was obtained and concentrated to 0.5 mL under a nitrogen stream. Subsequently, each residue was re-dissolved in 2 mL of aqueous methanol solution (5% methanol in water). Finally, the extracts were measured by the fluorescence signal amplification experiment.

Statistical Analysis
Fluorescence-emission spectra curves for AFM 1 were plotted using Origin 8.0 software (OriginLab Corporation, Northampton, MA, USA). Linear regression analysis of the fluorescence intensity as a function of the concentration of AFM 1 was carried out with Microsoft Excel. Each analysis including AFM 1 calibration curve standards and test samples was performed in triplicate. Standard deviations (SDs) and means of fluorescence intensity were determined from three replicates.

Design Strategy for AFM 1 Detection Based on a Graphene Oxide Sensing Platform
GO has many advantages due to its unique properties, including its great binding ability to single-stranded DNA (such as aptamers) through π stacking interactions between nucleobases and GO nanosheets and its high distance-dependent fluorescence quenching performance [35,39]. A GO-based aptasensor for the detection of AFM 1 was developed taking advantage of the above properties. A schematic illustration of the sensing platform is presented in Figure 1. In this sensing method, when the FAM-modified aptamer was incubated with the GO solution, the fluorescence signal quenched dramatically, demonstrating a strong binding between the aptamer and GO, with a high quenching efficiency. Upon the addition of AFM 1 , an AFM 1 /aptamer complex formed. Such an interaction can lead to a conformational change in the aptamer, causing a separation of the conjugated aptamer from the surface of GO. Thus, fluorescence is recovered, since GO would not be able to quench the fluorescence efficiently owing to the long distance. In order to confirm that the presence of AFM 1 can lead to the formation of an AFM 1 /aptamer complex and subsequently to fluorescence recovery, 10 ng mL −1 of AFM 1 was added to a Tris buffer solution that contained 200 nM of AFM 1 aptamer and 20 µg mL −1 of GO. As seen in Figure 2, a significant fluorescence enhancement was observed, demonstrating that the AFM 1 /aptamer complex was formed. More importantly, the covalently modified FAM had no impact on the recognition ability of the AFM 1 aptamer. high quenching efficiency. Upon the addition of AFM1, an AFM1/aptamer complex formed. Such an interaction can lead to a conformational change in the aptamer, causing a separation of the conjugated aptamer from the surface of GO. Thus, fluorescence is recovered, since GO would not be able to quench the fluorescence efficiently owing to the long distance. In order to confirm that the presence of AFM1 can lead to the formation of an AFM1/aptamer complex and subsequently to fluorescence recovery, 10 ng mL −1 of AFM1 was added to a Tris buffer solution that contained 200 nM of AFM1 aptamer and 20 μg mL −1 of GO. As seen in Figure 2, a significant fluorescence enhancement was observed, demonstrating that the AFM1/aptamer complex was formed. More importantly, the covalently modified FAM had no impact on the recognition ability of the AFM1 aptamer. DNase I was adopted as a signal amplification strategy to improve the sensitivity of the aptasensor. As shown in Figure 1, upon the addition of AFM1 and DNase I, the formation of the high quenching efficiency. Upon the addition of AFM1, an AFM1/aptamer complex formed. Such an interaction can lead to a conformational change in the aptamer, causing a separation of the conjugated aptamer from the surface of GO. Thus, fluorescence is recovered, since GO would not be able to quench the fluorescence efficiently owing to the long distance. In order to confirm that the presence of AFM1 can lead to the formation of an AFM1/aptamer complex and subsequently to fluorescence recovery, 10 ng mL −1 of AFM1 was added to a Tris buffer solution that contained 200 nM of AFM1 aptamer and 20 μg mL −1 of GO. As seen in Figure 2, a significant fluorescence enhancement was observed, demonstrating that the AFM1/aptamer complex was formed. More importantly, the covalently modified FAM had no impact on the recognition ability of the AFM1 aptamer. DNase I was adopted as a signal amplification strategy to improve the sensitivity of the aptasensor. As shown in Figure 1, upon the addition of AFM1 and DNase I, the formation of the AFM1/aptamer complex caused the dissociation of the aptamer conjugate from GO, and subsequently the aptamer was digested by DNase I. Once AFM1 was released from the DNase I was adopted as a signal amplification strategy to improve the sensitivity of the aptasensor. As shown in Figure 1, upon the addition of AFM 1 and DNase I, the formation of the AFM 1 /aptamer complex caused the dissociation of the aptamer conjugate from GO, and subsequently the aptamer was digested by DNase I. Once AFM 1 was released from the AFM 1 /aptamer complex, it was again available to bind to another aptamer, inducing a cyclic amplification of the fluorescence signal. As a consequence, a strong amplification of the fluorescence signal could be achieved for the quantification of AFM 1 .

Optimization of the Experimental Conditions
The concentration of GO would influence the fluorescence quenching efficiency. Therefore, to optimize the sensing platform, the effect of GO concentration on the change of the fluorescence signal was investigated. Various concentrations of GO were added to a solution containing 200 nM of AFM 1 aptamer. As seen in Figure S1, the fluorescence intensity decreased with increasing amounts of GO and reached the lowest level at a concentration of GO of 20 µg mL −1 . Thus, 20 µg mL −1 of GO solution was used for further sensing experiments.
To improve the signal amplification efficiency, the optimization of the concentration of DNase I was essential. In this experiment, we measured the fluorescence intensity of the complex with 10 ng mL −1 of AFM 1 . Various amounts of DNase I were added to the GO/aptamer solution containing 200 nM of AFM 1 aptamer and 20 µg mL −1 of GO. As seen in Figure S2, the fluorescence intensity increased as the DNase I concentration increased from 0 to 200 U, and the highest level of fluorescence was observed at 200 U of DNase I. In this case, the optimal amount of DNase I was determined as 200 U.

Analytical Performance of the Aptasensor
Under optimal conditions, the signal responses of the GO-based aptasensor to different concentrations of AFM 1 were measured using a DNase I-induced target recycling amplification platform. The fluorescence emission spectrum was measured for AFM 1 determination with excitation and emission wavelengths of 480 nm and 520 nm, respectively. As seen in Figure 3, the fluorescence intensity increased as the concentration of AFM 1 increased from 0.2 to 10 µg/kg. The calibration curve of fluorescence intensity versus AFM 1 concentrations was linear, as F = 65.77 C + 46.334 (R 2 = 0.9939), in which F is the fluorescence intensity, and C is AFM 1 concentration. The limit of detection of the amplification aptasensor was determined to be 0.05 µg/kg, which was calculated at the signal-to-noise ratio of 3. As shown in Table 1, the aptasensor displayed a sensitivity for AFM 1 comparable to those of other instrumental and rapid screening methods reported previously.
Sensors 2019, 19, x FOR PEER REVIEW 5 of 9 AFM1/aptamer complex, it was again available to bind to another aptamer, inducing a cyclic amplification of the fluorescence signal. As a consequence, a strong amplification of the fluorescence signal could be achieved for the quantification of AFM1.

Optimization of the Experimental Conditions
The concentration of GO would influence the fluorescence quenching efficiency. Therefore, to optimize the sensing platform, the effect of GO concentration on the change of the fluorescence signal was investigated. Various concentrations of GO were added to a solution containing 200 nM of AFM1 aptamer. As seen in Figure S1, the fluorescence intensity decreased with increasing amounts of GO and reached the lowest level at a concentration of GO of 20 μg mL −1 . Thus, 20 μg mL −1 of GO solution was used for further sensing experiments.
To improve the signal amplification efficiency, the optimization of the concentration of DNase I was essential. In this experiment, we measured the fluorescence intensity of the complex with 10 ng mL −1 of AFM1. Various amounts of DNase I were added to the GO/aptamer solution containing 200 nM of AFM1 aptamer and 20 μg mL −1 of GO. As seen in Figure S2, the fluorescence intensity increased as the DNase I concentration increased from 0 to 200 U, and the highest level of fluorescence was observed at 200 U of DNase I. In this case, the optimal amount of DNase I was determined as 200 U.

Analytical Performance of the Aptasensor
Under optimal conditions, the signal responses of the GO-based aptasensor to different concentrations of AFM1 were measured using a DNase I-induced target recycling amplification platform. The fluorescence emission spectrum was measured for AFM1 determination with excitation and emission wavelengths of 480 nm and 520 nm, respectively. As seen in Figure 3, the fluorescence intensity increased as the concentration of AFM1 increased from 0.2 to 10 μg/kg. The calibration curve of fluorescence intensity versus AFM1 concentrations was linear, as F = 65.77 C + 46.334 (R 2 = 0.9939), in which F is the fluorescence intensity, and C is AFM1 concentration. The limit of detection of the amplification aptasensor was determined to be 0.05 μg/kg, which was calculated at the signal-to-noise ratio of 3. As shown in Table 1, the aptasensor displayed a sensitivity for AFM1 comparable to those of other instrumental and rapid screening methods reported previously.

The Specificity of the Aptsensor
The specificity of the aptasensor was also investigated to assess the effect of other mycotoxins. The change of fluorescence intensity was measured under experiment conditions identical to those used for AFM 1 detection in the presence of four other mycotoxins (AFB 1 , OTA, ZEA and α-ZOL) at a concentration of 4 ng mL −1 . It can be seen that significantly higher fluorescence intensity was obtained in the case of AFM 1 determination in comparison with other mycotoxins and the control (Figure 4), which indicated that the specificity of this amplifying sensing platform is high for AFM 1 determination.

The Specificity of the Aptsensor
The specificity of the aptasensor was also investigated to assess the effect of other mycotoxins. The change of fluorescence intensity was measured under experiment conditions identical to those used for AFM1 detection in the presence of four other mycotoxins (AFB1, OTA, ZEA and α-ZOL) at a concentration of 4 ng mL −1 . It can be seen that significantly higher fluorescence intensity was obtained in the case of AFM1 determination in comparison with other mycotoxins and the control (Figure 4), which indicated that the specificity of this amplifying sensing platform is high for AFM1 determination.

Method Validation
Ultimately, the applicability and reliability of the aptasensor platform were evaluated by detecting different concentrations of AFM1 in infant milk powder samples. As indicated in Table 2, the recovery of the spiked infant milk powder samples ranged from 92% to 126%, demonstrating that the amplification strategy developed in this work can be useful as a quantitative method for AFM1 analysis in real samples for food safety.

Method Validation
Ultimately, the applicability and reliability of the aptasensor platform were evaluated by detecting different concentrations of AFM 1 in infant milk powder samples. As indicated in Table 2, the recovery of the spiked infant milk powder samples ranged from 92% to 126%, demonstrating that the amplification strategy developed in this work can be useful as a quantitative method for AFM 1 analysis in real samples for food safety.

Conclusions
A novel graphene oxide-based aptasensor was developed for the detection of AFM 1 with high sensitivity and specificity. This technique uses the properties of GO as an aptamer protector against nuclease cleavage, thereby allowing DNase I to cleave the aptamer for a target cycling signal amplification. Under the optimal conditions, a good linear relationship was detected between fluorescence intensity and AFM 1 levels in the range of 0.2 to 10 µg/kg, with a detection limit of 0.05 µg/kg. Satisfactory recoveries were measured in infant milk powder samples spiked with different concentrations of AFM 1 . Furthermore, the aptasensor proposed in this work is rapid, simple and low-cost in comparison with other methods reported previously. This study could thus provide a very promising platform for the analysis of AFM 1 in dairy products. More importantly, the aptasensor could be improved by replacing aptamer sequences for the detection of other food safety targets.

Author Contributions:
This work proposed in this paper was carried out in collaboration with all the authors. X.G. and F.W. proposed the idea of the paper, wrote the original paper and analyzed the experimental data. Q.Q. and N.Z. supported the structure of the paper. M.S. and M.-L.F. and J.W revised the paper.