**4. Fluorometric aptamer**

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 method was 0.05 nM.

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

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

fluorophores of two molecules and achieve enzyme-free amplification and significantly improve the signal-to-background ratio and sensitivity of AFB1 detection.

Lu et al. [27] discovered another interesting fluorescence method. Their experiments reported a target-driven switch-on fluorescence aptasensor for monitoring AFB1 determination by employing the fluorescence resonance energy transfer (FRET) between the CdZnTe quantum dots (QDs) and Au nanoparticle (AuNP) pair. AuNPs is considered to be one of the most widely used metal NPs. It can promote electron transfer and act as a tiny conduction center. The crucial design of this switch is that the AuNP acceptors were bioconjugated with the thiol groupmodified complementary DNA (cDNA) of aptamer. In this case, as the CdZnTe QDs (energy donor) approaches AuNPs(energy acceptor), FRET is produced, leading to the subsequent fluorescence disappearance of CdZnTe QDs, while AFB1 specifically binds to the aptamer, and aptamer breaks away from AuNPs. Thus, CdZnTe QDs separates from AuNPs, leading to the subsequent fluorescence recovery of CdZnTe QDs. This aptasensor is simple in design and has the advantages of wide linear range, low LOD, high sensitivity, and selectivity.

Wang et al. [28] synthesized a novel fluorescent nitrogen-doped carbon quantum dot (N, C-dots) and combined it with the aptamer/AuNP complex for detection of AFB1. Initially, they synthesized a positively charged fluorescent N, C-dots by hydrothermal treatment of trypsin, synthesized AuNP by a typical citrate reduction method, and attached a thiol-labeled oligonucleotide (AFB1 aptamer) to AuNP. N, C-dots/aptamer/AuNP nanocomposite is formed on the surface. N, C-dots are mainly used as a quencher for the construction of aptamer sensors. When AFB1 is absent, N, C-dots bind to aptamer/AuNPs by electrostatic interaction, and the fluorescence of N, C-dots is quenched by AuNPs. When AFB1 is present, the aptamer binds to AFB1, N, C-dots are released, and its fluorescent signal is restored. Therefore, by measuring the fluorescent signal of N, C-dots, the concentration of AFB1 can be obtained. The detection system is extremely sensitive with a detection limit of 5 pg/mL (16 pM).

Beheshti-Marnani et al. [13] developed aptasensor assembled with assisting reduced graphene oxide nanosheets as the signal amplifier was fabricated and applied for detecting ultralow levels of AFB1 through a nanobiology interaction system. The detection principle and procedures are different from fluorescence method; the steps are as follows: (1) synthesis of reduced graphene nanosheets (rGO), (2) fabrication of the AFB1 aptasensor, (3) immobilizing AFB1 binding ssDNA aptamer on the surface of electrode, and (4) cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) characterized by the modified glassy carbon electrodes. Differential pulse voltammetry (DPV) was used to quantitatively analyze aflatoxin B1 in practical samples. This new technology is characterized by its simplicity, low cost, and sensitive label-free, in particular, the ability to detect very small quantities of aflatoxin B1 with a considerable low limit of detection (LOD = 0.07 nM) and good repeatability (RSD = 2.9) and stability.

Wang et al. [29] report a versatile ratiometric fluorescence platform for multiple detection of various targets based on the conjugation of single-stranded DNA (ssDNA) with protonated graphitic carbon nitride nanosheets (Pg-C3N4NSs). This method is also feasible for AFB1; the principle is that Pg-C3N4NSs promotes oxidation of substrate o-phenylenediamine (OPD) by binding to ssDNA in the presence of H2O2. Subsequently, the fluorescence signal at 564 nm of the oxidation product 2,3-diaminophenazine (DAP) was collected and concurrently quenches the intrinsic fluorescence of conjugates ssDNA/Pg-C3N4 NSs at 443 nm upon excitation at 370 nm. Lastly, the transformation of fluorescence was used for ratiometric fluorescence-based analytical. This method applies to for multiplex detection of various targets.

**99**

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

Wu et al. [30] proposed a simple electrochemical body sensor, and they take advantage of host-guest identification between ferrocene and β-cyclodextrin (β-CD) to detect AFB1. Despite the long-time consumption and complexity involved in the preparation process of the pβ-CD/AuNPs/GC electrode and AFB1-sensitive dsDNA, they demonstrated the selectivity, stability, and reproducibility of the electrochemical aptasensor in the detection of AFB1; there is no significant difference in stability between 1 day and 15 days, that is to say, electrochemical aptasensor has

Abnous et al. [31] built an electrochemical biosensor for accurate detection of AFB1. AFB1 is based on aptamer to form a π-shape complementary strand of aptamer (CSs) complex on the surface of electrode and exonuclease I (Exo I). The purpose of π-shape design is to greatly increase the sensitivity of aptamer. In the absence of AFB1, the PI configuration of the gold electrode surface remains intact, and a double potential barrier is formed on the electrode surface, limiting the

cal signals are measured. When AFB1 exists,π-shape structure was removed, and a strong current was recorded after the addition of Exo I. Under the optimum conditions, the concentration range of AFB1 can be detected in the range of 7–500 pg/mL

Xia et al. [32] designed a new split-type photoelectrochemical (PEC) immunosensing platform for sensitive detection of AFB1, combined with the etching reaction triggered by the enzymatic hydrolysis of cobalt oxyhydroxide (CoOOH) at the functional interface of cadmium sulfide (CdS) nanoparticles. The concentration of CdS nanoparticles has a great influence on the analytical properties of PEC biosensor. Excessive CdS may induce high background signal, while low concentration produces weak photocurrent response. In their experiment, the optimum concentration of CdS nanoparticles was 0.8 mg/mL–1, and the entire time of the method is within 1.5 h for each sample. Under optimal conditions, the detection

Li et al. [33] developed an aptamer structure switch experiment with horseradish peroxidase (HRP) labeling for sensitive absorbance and chemiluminescence detection of small molecules. Differently from competitive enzyme-linked immunosorbent assay (ELISA), they fixed the cDNA of the aptamer to the surface of the

Zhao et al. [34] developed a novel nano-antibody and magnetic beads-based directed competitive ELISA (MB-dcELISA) based on both recombinant antibody and its mimotope for AFB1 detection. The 50% inhibition concentration and detection limit of MB-dcELISA were 0.75 and 0.13 ng/mL, respectively, and the linear

Zhang et al. [35] discovered a novel anti-AFB1 monoclonal antibody in order to establish a sensitive immunoassay for AFB1, and a novel CdTe/CdS/ZnS quantum dot fluorescence probe was synthesized by binding to the surface of CdTe/CdS/ZnS

<sup>4</sup><sup>−</sup> with the electrode surface, and only weak electrochemi-

, and the accuracy of this method (expressed in

**5. Electrochemical aptamer**

good stability.

contact of [Fe(CN)6]

3−/

and a limit of detection (LOD) of 2 pg/mL.

limit of this method is 2.6 pg/mL<sup>−</sup><sup>1</sup>

**6. Aptamers with chemiluminescence immunoassay**

RSD) is ±8.6%.

microporous plate.

range was 0.24–2.21 ng/mL.

**7. Others**
