According to the statistics, about 25% of the agricultural products are contaminated with mycotoxins during crop growth, storage or processing in the world [1]. Fungal contamination of food is a global concern, most of the toxic substances produced by pollution have strong carcinogenic effects on human beings [2]. Aflatoxin is the most important and toxic mycotoxin among many mycotoxins and AFB1 is the most toxic aflatoxin. Many crops such as peanuts, corn and grains are vulnerable to AFB1 contamination, which makes AFB1 a serious threat to food safety and human health [3]. At present, more than 100 countries and regions have set limits for AFB1 in various foods ranging from 1 to 20 µg/kg [4]. Therefore, there is an urgent need to establish a fast, safe, accurate and capable of on-site detection of AFB1 method.
At present, the methods used for AFB1 detection mainly include thin layer chromatography (LC), high-performance liquid chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC-MS) [4]. Although these methods have good accuracy and high sensitivity, they have disadvantages such as tedious sample pretreatment process, expensive instruments, highly trained professionals and so on, which limit their application for rapid and on-site screening of AFB1 [5]. Due to these disadvantages of traditional methods, fluorescence detection which has the advantages of high sensitivity, simple operation and simultaneous detection of multiple toxins, has attracted the attention of researcher [6]. With the rapid development in recent decades, various fluorescent biosensors have emerged in an endless stream [7]. In order to enhance the specific recognition of the sensor and the target, aptamer as target recognition element compared with antibody, enzyme, etc.
Aptamer has stronger affinity with the target, the simpler and cheaper synthesis process and better stability. What’s more, the aptamer is not easy to be limited by the storage environment, temperature, and its resistance to degeneration [8]. Li et al. reported a new aptamer fluorescence sensor, whcih can sensitively detect Pb2+ [9]. Yang et al designed a novel fluorescence based protein detection method using aptamer probe as recognition molecule and cationic conjugated polymer (CCP) as reporter molecule [10]. At present, the fluorescence aptasensors prepared by using aptamer instead of antibody has been widely used in the detection of mycotoxins, protein, cell [11].
Most of the fluorescence aptasensors require a tedious and time-consuming process of embedding the probe or some recognition molecules on the surface of the material used to prepare the sensor, which will greatly increase the preparation time of the sensor. In addition, the separation and purification process involved in fixing the probe and identifying the molecules will lead to the signal damage of the sensor and affect the performance of the sensor. To solve this problem, many researchers have chosen to use homogeneous sensors [12–13], which is kept in a solution from preparation to detection without any separation operation. Fluorescence resonance energy transfer (FRET) is a commonly used method for quenching fluorescent dyes in fluorescence sensors. The FRET process requires a distance between the quenching material and the fluorescence probe within 10 nm. When the quenching material and the fluorescence probe are dispersed in the system, the FRET process is weak. It almost has no effect on the signal of fluorescent probe, which is convenient for the application of homogeneous detection in fluorescent adaptor sensor. Lu[14] et al. provided a simple, homogeneous and sensitive strategy mediated FRET for the histone analysis. The homogeneous sensor has been widely used in the detection of mycotoxins, protein, cell[15].
Furthermore, the conventional fluorescent aptasensors were designed based on the changes of the “signal on” or “signal off” of single probe. The conventional fluorescent aptasensors were easily affected by the environmental and instrument factors in detection of the actual sample, resulting in the degradation of the sensor performances[16]. Yang et al.[17] prepared a simple and rapid fluorescence homogeneous sensor for AFB1 detection by using the adaptation system of BHQ2-labeled complementary chain and Cy3 labeled fluorescent probe. However, the signal ratio is not sensitive to changes affected by environmental and instrumental. To improve the stability and accuracy of the sensor, the ratiometric fluorescent aptasensor was selects two signal probes with different excitation and emission wavelengths. The final signals are determined by the fluorescence signal ratio of the two probes [18–19]. Therefore, the ratiometric fluorescent aptasensor using two signal probes has become a new research hotspot.
In this paper, a simple, rapid and sensitive ratio homogeneous fluorescent aptasensor was constructed by using aptamer with hairpin structure labeled simultaneously with Cy5, BHQ2 and cDNA labeled with Cy3 to detect AFB1. The AFB1 aptamer with hairpin structure was used as recognition element, and the labeled BHQ2 whcih can quench the fluorescent dyes Cy3 and Cy5. The aptamer labeled with Cy5 and BHQ2 was paired with the cDNA labeled with Cy3 to form a double strand through complementary pairing. At this time, Cy3 was close to BHQ2, leading to fluorescence quenching. A high fluorescence emission of Cy5 emerged due to the distance of BHQ2. The hairpin structure would be opened in the presence of AFB1.The fluorescence intensity of Cy3 was increased due to it was far away from BHQ2, and the fluorescence intensity of Cy5 was decreased due to Cy5 approaching BHQ2. AFB1 was detected by comparing the ratio of FCy3 to FCy5 before and after adding AFB1.