Enzyme-free amplified and ultrafast detection of aflatoxin B1 using dual-terminal proximity aptamer probes
Introduction
Aflatoxin B1 (AFB1) is considered as one of the most harmful toxins and has been classified as a group I carcinogen by the International Agency for Research on Cancer (Wu, 2011). Thus, AFB1 contamination is the critical problem of food safety (Turner, Subrahmanyam, & Piletsky, 2009). At present, methods for the analysis of AFB1 are mainly based on two categories: chromatography (Huertaspérez et al., 2018, Var et al., 2007) and immunoassay (Duan et al., 2016). Although chromatography methods are highly accurate tools for AFB1 detection, they suffer from the demands of sophisticated instruments and time-consuming procedures. Meanwhile, immunoassay method enables rapid and sensitive detection of AFB1 (Lin et al., 2018, Tang et al., 2018). Yet, it is reliant on antibodies which are costly and difficult to preserve (Dunn et al., 2017, Wang et al., 2013). Besides, it usually involves separation steps which would complicate the detection process (Duan et al., 2016).
Aptamer-based assay has aroused great concern (Luo et al., 2015, Ma et al., 2015, Xia et al., 2018, Xia et al., 2018, Zhou et al., 2016), and has been emerged as an alternative method to immunoassay for detection of food contaminants, such as AFB1 (Chen et al., 2017, Duan et al., 2016, Sabet et al., 2017, Taghdisi et al., 2018, Wang et al., 2019). Aptamers (DNA or RNA) are specific affinity reagents that can be acquired by in vitro selection (Huang et al., 2015; Wang et al., 2017). Benefited from the nucleic acid nature of the controllability, designability and high stability (Deng et al., 2017, Deng et al., 2018, Dunn et al., 2017), aptamers have been designed to construct various detection methods (Meng et al., 2016, Wang et al., 2011, Wu et al., 2018, Wu et al., 2015). Especially, the most prominent advantage of aptamers lies in that they can be engineered into numerous structure-switching oligonucleotide probes to directly output signals, such as aptamer molecule beacons or antisense displacement probes (Nakatsuka et al., 2018, Porchetta et al., 2012, Ricci et al., 2016), which can be used to construct the homogeneous assay (Dunn et al., 2017). The homogeneous assay would accommodate the elimination of the complex separation process and result in one-tube test with more efficient and quick recognition of target molecules.
In general, however, one target can usually trigger only one signal output (usually fluorescence signal) using structure-switching aptamer probes (Dunn et al., 2017), which may result in relatively low sensitivity. Thus, amplification strategies have been adopted to amplify the signal, such as rolling circle amplification (Yang, Fung, Cho, & Ellington, 2007) and RT-qPCR (Guo et al., 2014). Undoubtedly, these amplification strategies can significantly improve the detection sensitivity of aptamer probes. Nevertheless, these amplification processes usually need complex enzyme catalysis steps with the involvement of enzymes and precise temperature control, hindering their application in on-spot detection of AFB1.
In this work, we propose a general design strategy for constructing aptamer probes, enabling enzyme-free amplified, ultrafast and one-tube homogenous detection of AFB1. The aptamer probes are designed with dual-terminal proximity structures, allowing to light up of two fluorophores upon binding with one molecule target AFB1, leading to enzyme-free amplification and remarkable enhancement of signal to background ratio, in turn improving the sensitivity of structure-switching aptamer probes. Besides, benefiting from the high affinity of aptamers and homogeneous reactions, this aptamer probe can quickly and specifically response to AFB1, and the detection process of AFB1 could be completed within 1 min. The proposed aptamer probe has been successfully applied to detect AFB1 in the peanut oil and broad bean paste. The sensing procedures are ultrafast and simple, and can be conducted in one-test tube at constant temperature, eliminating complex separation process. Contributed to the generality and simplicity of the design strategy, this structure-switching probe would act as a general platform for rapid, sensitive and on-spot detection of food contaminants.
Section snippets
Materials and reagents
Aflatoxin B1, aflatoxin B2, aflatoxin M1, ochratoxin A and zearalenone were bought from Fermentek Ltd. (Jerusalem, Israel). All DNA sequences (Table S1) were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). Labeled sequences were HPLC purified, and other sequences were PAGE purified. The sequence of AFB1 binding aptamer was 5′-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3′ (single stranded DNA oligonucleotides with 50 nucleotides),
Principle of dual-terminal proximity aptamer probes
The most intriguing feature of dual-terminal proximity aptamer probes is that it could achieve enzyme-free amplified, rapid and specific recognition of target AFB1 (Scheme 1). It has been designed with a mixture of double-stranded and single-stranded structure, possessing two stems at 5′ and 3′ terminal, and one loop in the middle region. The aptamer probe can be finely tuned with different stability by changing the number of complementary bases on each stem, allowing elaborately optimize probe
Conclusions
In this work, we successfully construct an enzyme-free amplified, ultrafast, one-tube and homogeneous method for AFB1 analysis by designing a dual-terminal proximity structured aptamer probe. The prominent advantages of the aptamer probe lie in its enzyme-free amplified strategy (one molecule could trigger two signal outputs) and ultrafast recognition process. The dual-terminal proximity structure could lead to enzyme-free amplification and remarkable improvement of the signal to background
Acknowledgements
This work was financially supported by National Natural Science Foundation of China (No. 21804095, No. 51773129), China Postdoctoral Science Foundation (No. 2018M631079) and the Fundamental Research Funds for the Central Universities (No. 2018SCU12048, No. 1083304121001).
Conflict of interest
The authors have declared no conflict of interest.
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These authors contributed equally to the work.