A New Approach for Detection of Aflatoxin B1

*Xing-Zhi-Zi Wang*

### **Abstract**

Aflatoxin B1 (AFB1) is harmful to human health, mainly resulting from its toxic effects on the liver. AFB1 can lead to liver cell necrosis, hemorrhage, fibrosis, cirrhosis, etc. Acute AFB1 exposure at high levels can lead to hepatitis, whereas chronic exposure can result in liver cancer. In the past decades, a series of methods and techniques for detecting AFB1, including enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC), have been developed. This study reviewed the detection methods of AFB1 and the corresponding utilization and summarizes all methods for evaluating the toxification of AFB1.

**Keywords:** aflatoxin B1, purification, detection, aptasensor, biosensor, reduced graphene nanosheets

### **1. Introduction**

Aflatoxin B1 (AFB1) is mainly a metabolite produced by *Aspergillus flavus*. AFB1 poses a threat to human health due to its three huge toxicities [1]. The toxic effects are as follows [2]. First, it is genetically toxic and can cause DNA damage. Second, aflatoxin shows strong hepatophilic properties when it enters the human body and can cause liver cell necrosis, hemorrhage, fibrosis, and cirrhosis. Finally, aflatoxin has high toxicity and strong carcinogenicity. The data show that its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. The carcinogenic force is 70 times that of the known carcinogen dimethyl nitrosamines and 900 times that of butter yellow (methyl azobenzene) [3]. The carcinogenic pathway is mainly activated by cytochrome p450 (CYP) monooxygenase system, and AFB1 is metabolized by CYP1A2 and CYP3A4 to produce epoxy compounds, including active epoxy resins (aflatoxin-8,9-epoxy, AFBO), which generate mutagenic aflatoxin-n7-guanine adduct (AFB1-N7-gua) through interaction with DNA and cause DNA damage to varying degrees [4]. Since AFB1 is toxic to the human body, it is necessary to monitor the content of AFB1 in food. However, AFB1 pollution still exists in a small number of remote areas due to poor living standards and quality. Considering the feasibility and economic feasibility of AFB1 detection technology, the government needs to add feasible, fast, and accurate new technical schemes for supervision [5]. Therefore, it is of great significance to study the new progress in the detection of AFB1 in food.

Detection of AFB1 is divided into two processes, including purification of AFB1 and quantitative analysis of AFB1. Purification methods of AFB1 mainly include liquid–liquid extraction, dispersive liquid–liquid microextraction, solid-phase

extraction, molecularly imprinted polymer, immunoaffinity column, etc. [6]. The quantitative analysis methods of AFB1 mainly include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), etc. These methods are tedious, time-consuming, and expensive; moreover, the sample processing is complex and requires professional operation, which is not suitable for the rapid and effective detection and analysis of AFB1. Therefore finding a fast and sensitive method has important application value.

With the rapid development of science and technology, scientists have been concerned about inventing a fast and sensitive method to detect AFB1. AFB1 detection based on aptamer AFB1 sensor is the most widely used detection technology. The aptamer is a single-stranded nucleic acid or peptide molecule; it has a unique secondary structure and can specifically bind to the target, like proteins, drugs, and other biomolecules [7]. Aptamer-based biosensors (aptasensors) have been widely used owing to high sensitivity, selectivity, accuracy, fast response, and low cost [8–10]. Fluorescent-based optical biosensors are the most commonly used method. Combining fluorescent pigment molecules with fluorescent aptasensors leads to the generation of light in the process of biological recognition interaction, so as to achieve the detection of target molecules [11]. In addition, nanomaterials have been widely used in biomolecular detection, such as graphitic carbon nitride nanosheets (g-C3N4 NSs) [12] and reduced graphene nanosheets (rGO) [13].

#### **2. Purification of aflatoxins**

The purification is the key step in the detection of the level of AFB1; traditional methods of AFB1 mainly include liquid–liquid extraction (LEE), dispersive liquid–liquid microextraction, solid-phase extraction (SPE), molecularly imprinted polymer, immunoaffinity column, etc.; most of these are time-consuming and expensive [14]. Encouragingly, Xie J et al. [6] provide the first report of a broadspectrum specific mAb-modified reduced graphene nanosheets (rGO) film that can be designed to extract and purify AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 in rabbit serum. This method is suitable for analysis of different types of analyses from different samples. Compared with the traditional method, this method has the advantages of high selectivity, simplicity, low sample consumption, and the use of a small amount of organic solvent, especially extraction of ultra-trace levels of AFs.

However, in the process of extracting AFB1, the complexity of food components, especially fat, causes some interference to AFB1. In addition, AFB1 is lipophilic; it is difficult to extract AFB1 from soybean and vegetable oil [14, 15]. The purification is not strong enough, the AFB1 in vegetable oil cannot be completely removed, and lower lever of AFB1 will also lead to human liver damage [1, 16]. Xi Yua et al. analyzed trace amounts of AFB1 in vegetable oils by combining LTC and immuno-magnetic solid-phase extraction (IMSPE) with fluorescence spectroscopy (FL) detection. This process removed fat interference in vegetable oil samples. Subsequently, IMSPE enhances the selectivity and efficiency of extraction through specific antibody–antigen binding. The advantage of this method is that the combined application of traditional LTC and modern IMSPE improves the sensitivity and selectivity of extraction process and meanwhile reduces the time and cost.

#### **3. Application of aptamer**

Nucleic acid aptamers are single-stranded oligonucleotides screened in vitro by systematic evolution of ligands by exponential enrichment (SELEX), which are

**97**

*A New Approach for Detection of Aflatoxin B1 DOI: http://dx.doi.org/10.5772/intechopen.90403*

**4. Fluorometric aptamer**

method was 0.05 nM.

A, but it is more likely to spread to other toxins.

widely concerned as a new biometrics. SELEX technology can be used to screen the combination of the target molecule specific adaptor and target specific [17]. Thus the aptamer has the characteristics of simple preparation, strong specificity, good stability, and a very wide range of target substances, including analysis and detection, biochemistry, food safety [18], clinical medicine [19], and other fields [20]. According to the design principles in different fields, adaptors can be converted into different signals. The commonly used ligand biosensors include fluorescence adaptor sensor, colorimetric adaptor sensor, electrochemical adaptor sensor, etc. In recent years, the aptamer has been applied to the detection of AFB1, which has greatly improved the detection efficiency and sensitivity of AFB1 in the field of aflatoxin sensor construction; according to various researches at home and abroad, electrochemical biosensors have been constructed with antibody, enzyme and nucleic acid aptamer as recognition elements; and enzyme catalysis technology, DNA self-assembly technology, ionic liquid, nano materials, conductive polymer have been used to metal compounds, etc. for the detection of aflatoxin [21–23].

Ye et al. [24] developed a low-cost, high-sensitivity fluorescence polarization (FP) assay by using GO-based fitness biosensors to detect AFB1. Fluorescein amidite (FAM) labeled the aptamers fitness combines with the surface of GO to form the aptamer/GO macromolecular complex. In the presence of AFB1, the opposite dissociates from the GO surface and binds to AFB1 specifically to form the aptamer/ AFB1 complex. As a result, large changes in the molecular weight of the aptamer were observed before and after the combination, leading to significant changes in the fluorescence polarization (FP) value. The lowest detection limit (LOD) of this

Li et al. [25] use a fluorometric aptamer-based method to detect the level of aflatoxin B1 (AFB1). Their assay aims to develop a simple and sensitive label-free fluorescence aptasensor to monitor and control AFB1 in foodstuffs quickly and accurately. In their experiment, the AFB1 aptamer with the fluorescent dye thioflavin T (ThT) forms a AFB1 aptamer/ThT G-quadruplex complex in the absence of AFB1, increasing the fluorescence intensity of ThT. While the AFB1 aptamer with AFB1 forms a AFB1 aptamer/AFB1 complex in the presence of AFB1, causing the fluorescence intensity to decrease, the levels of AFB1 were directly correlated to fluorescence intensity. The general experimental procedures are as follows: first of all, the samples were preprocessed; then, the experimental conditions were optimized, including the optimum ratio of AFB1 aptamer: ThT, the concentration of KCL and the reaction time (20 min); lastly, using a LUMINA Fluorescence Spectrometer, the fluorescence intensity at excitation/emission wavelengths of 440 nm/487 nm was tested. In this case, the results were in good agreement with those obtained from commercial ELISA kits; the advantages of this method are simpler and more convenient—no label, low cost, and higher efficiency and specificity. The more evidence [8] has proven that this fluorometric aptamer-based method has great practical applications in food industry; not only does it detect AFB1 and ochratoxin

Xia et al. [26] designed a dual-terminal proximity structured aptamer probe; the main purpose of this design is to construct an enzyme-free, ultrafast, single-tube, homogeneous AFB 1 analysis method. This aptamer probe can quickly respond to AFB1, and the detection process can be completed within 1 min, which is one of the fastest detection methods for AFB1. Aptamer probe is the design to dual-terminal proximity structures, which allows the binding of one molecule to illuminate the

#### *A New Approach for Detection of Aflatoxin B1 DOI: http://dx.doi.org/10.5772/intechopen.90403*

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

extraction, molecularly imprinted polymer, immunoaffinity column, etc. [6]. The quantitative analysis methods of AFB1 mainly include enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), etc. These methods are tedious, time-consuming, and expensive; moreover, the sample processing is complex and requires professional operation, which is not suitable for the rapid and effective detection and analysis of AFB1. Therefore finding a fast and sensitive method has important application value. With the rapid development of science and technology, scientists have been concerned about inventing a fast and sensitive method to detect AFB1. AFB1 detection based on aptamer AFB1 sensor is the most widely used detection technology. The aptamer is a single-stranded nucleic acid or peptide molecule; it has a unique secondary structure and can specifically bind to the target, like proteins, drugs, and other biomolecules [7]. Aptamer-based biosensors (aptasensors) have been widely used owing to high sensitivity, selectivity, accuracy, fast response, and low cost [8–10]. Fluorescent-based optical biosensors are the most commonly used method. Combining fluorescent pigment molecules with fluorescent aptasensors leads to the generation of light in the process of biological recognition interaction, so as to achieve the detection of target molecules [11]. In addition, nanomaterials have been widely used in biomolecular detection, such as graphitic carbon nitride nanosheets

(g-C3N4 NSs) [12] and reduced graphene nanosheets (rGO) [13].

The purification is the key step in the detection of the level of AFB1; traditional methods of AFB1 mainly include liquid–liquid extraction (LEE), dispersive liquid–liquid microextraction, solid-phase extraction (SPE), molecularly imprinted polymer, immunoaffinity column, etc.; most of these are time-consuming and expensive [14]. Encouragingly, Xie J et al. [6] provide the first report of a broadspectrum specific mAb-modified reduced graphene nanosheets (rGO) film that can be designed to extract and purify AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 in rabbit serum. This method is suitable for analysis of different types of analyses from different samples. Compared with the traditional method, this method has the advantages of high selectivity, simplicity, low sample consumption, and the use of a small amount of organic solvent, especially extraction of ultra-trace levels of AFs. However, in the process of extracting AFB1, the complexity of food components,

especially fat, causes some interference to AFB1. In addition, AFB1 is lipophilic; it is difficult to extract AFB1 from soybean and vegetable oil [14, 15]. The purification is not strong enough, the AFB1 in vegetable oil cannot be completely removed, and lower lever of AFB1 will also lead to human liver damage [1, 16]. Xi Yua et al. analyzed trace amounts of AFB1 in vegetable oils by combining LTC and immuno-magnetic solid-phase extraction (IMSPE) with fluorescence spectroscopy (FL) detection. This process removed fat interference in vegetable oil samples. Subsequently, IMSPE enhances the selectivity and efficiency of extraction through specific antibody–antigen binding. The advantage of this method is that the combined application of traditional LTC and modern IMSPE improves the sensitivity and selectivity of extraction process and meanwhile reduces the time and cost.

Nucleic acid aptamers are single-stranded oligonucleotides screened in vitro by systematic evolution of ligands by exponential enrichment (SELEX), which are

**2. Purification of aflatoxins**

**3. Application of aptamer**

**96**

widely concerned as a new biometrics. SELEX technology can be used to screen the combination of the target molecule specific adaptor and target specific [17]. Thus the aptamer has the characteristics of simple preparation, strong specificity, good stability, and a very wide range of target substances, including analysis and detection, biochemistry, food safety [18], clinical medicine [19], and other fields [20]. According to the design principles in different fields, adaptors can be converted into different signals. The commonly used ligand biosensors include fluorescence adaptor sensor, colorimetric adaptor sensor, electrochemical adaptor sensor, etc. In recent years, the aptamer has been applied to the detection of AFB1, which has greatly improved the detection efficiency and sensitivity of AFB1 in the field of aflatoxin sensor construction; according to various researches at home and abroad, electrochemical biosensors have been constructed with antibody, enzyme and nucleic acid aptamer as recognition elements; and enzyme catalysis technology, DNA self-assembly technology, ionic liquid, nano materials, conductive polymer have been used to metal compounds, etc. for the detection of aflatoxin [21–23].
