Engineering DNA G-quadruplex assembly for label-free detection of Ochratoxin A in colorimetric and fluorescent dual modes

Highlights

• Colorimetric and fluorescent dual modes have been achieved for OTA sensing.

• An adenine nucleotide boosted the peroxidase activity of G-quadruplex-hemin.

• Engineering G-quadruplexes for signal amplification greatly simplified the assay.

Abstract

Colorimetric and fluorescent methods for Ochratoxin A (OTA) detection are convenient and well received. However, the pigments and autofluorescence originated from food matrices often interfere with detection signals. We have developed a strategy with colorimetric and fluorescent dual modes to solve this challenge. In the colorimetric mode, OTA aptamer (AP9) was assembled into a DNA triple-helix switch with a specially designed signal-amplifying sequence. The OTA-induced G-quadruplex (G4) of AP9 would open the switch and release the signal-amplifying sequence for colorimetric signal amplification. The G4 structures of AP9 were further utilized to combine with the fluorogenic dye ThT for fluorescent mode. By skillfully engineering DNA G4 assembly for signal amplification, there was no need for any DNA amplification or nanomaterials labeling. Detections could be carried out in a wide temperature range (22–37 ℃) and finished rapidly (colorimetric mode, 60 min; fluorescent mode, 15 min). Broad linear ranges (colorimetric mode, 10–1.5 ×10³ μg/kg; fluorescent mode, 0.05–1.0 ×10³ μg/kg) were achieved. The limit of detection for colorimetric and fluorescent modes were 4 μg/kg and 0.01 μg/kg, respectively. The two modes have been successfully applied to detect OTA in samples with intrinsic pigments and autofluorescence, showing their applicability and reliability.

Introduction

Ochratoxin A (OTA), produced by some species of fungi Penicillium and Aspergillus, occurs widely in cereal-derived products, dried fruits, spices, oil, and wine (Kumar et al., 2020). OTA is extremely stable even in highly processed foods and has a long half-life in human serum (Jiang et al., 2018, Kumar et al., 2020). The excess exposure of OTA causes serious risks, such as teratogenicity, carcinogenicity, hepatotoxicity, immunotoxicity, and nephrotoxicity (Kumar et al., 2020, Tao et al., 2018). Furthermore, the International Agency for Research on Cancer has classified OTA as a possible human carcinogen (group 2B) (Kumar et al., 2020). Consequently, maximum tolerated OTA levels have been strictly established by several countries and regions (Yang et al., 2011). Rapid and accurate monitoring of OTA is crucial for avoiding the risk of OTA exposure (Alhamoud et al., 2019, Ding et al., 2020, Jiang et al., 2018, Qian et al., 2020, Wang et al., 2017, Yang et al., 2011, Zhu et al., 2021). Analysis of OTA is usually performed by chromatographic methods (Giovannoli et al., 2014, Valenta, 1998), such as high-performance liquid chromatography (HPLC) coupled with fluorescence or mass spectrometric detector. However, chromatographic methods require sophisticated equipment and very strict sample pretreatment, resulting in high costs in manpower, equipment and time. Recently, immunoassays, based on antigen-antibody interactions, are emerging as simple and rapid methods for OTA analysis (Liu et al., 2008, Yu et al., 2005). However, the preparation of antibodies for mycotoxins is expensive and challenging due to the low antigenicity of OTA. Besides, the storage and application conditions of the antibody such as the temperature, pH, and ionic strength, are rigidly specified, which limit their practical applications.

In recent years, the flourishing development of novel materials and nanotechnologies has provided the accessibility of many useful chemo/biosensors (He et al., 2019, Liu et al., 2019, Lv et al., 2018, Nguyen and Kim, 2020, Su et al., 2021, Wongkaew et al., 2019). Among them, optical sensors generating colorimetric or fluorescent signals have drawn widespread attention (Nguyen and Kim, 2020, Su et al., 2021). Because their signals are easy to be observed by the naked eye and captured by digital camera or smartphone (Chen et al., 2021, Chen et al., 2021, He et al., 2012, Majdinasab et al., 2020, Yang et al., 2020a, Yang et al., 2020b), colorimetric and fluorescent detection methods are becoming increasingly popular, especially in resource-limited countries and regions. Innovative colorimetric and fluorescent (bio)sensors have been reported for the rapid and sensitive detection of OTA (Jia et al., 2021, Li et al., 2019, Li and Zhao, 2019, Tang et al., 2020, Zhu et al., 2020). Among these, DNA (bio)sensors are especially noteworthy (Li et al., 2019, Li and Zhao, 2019). Due to their easy synthesis, high stability and cost-effectiveness, DNA sensors have been elaborately constructed for OTA detection with colorimetric or fluorescent signal output. In some cases, the sensing of OTA were converted to the detections of DNA, thus it would be much easier to transduce and amplify signals through DNA amplification. In other instances, bioenzymes and/or nanomaterials, such as horseradish peroxidase (HRP), gold nanoparticles (AuNPs), quantum dots (QDs), were labeled to amplify signals for sufficient sensitivity (Du and Dong, 2017, Ma et al., 2016). These designs were ingenious, but the amplification of DNA and the preparation of bioenzyme and/or nanomaterials for signal amplification involved multi-step procedures, increased cost obviously. Besides, due to the intrinsic pigments and autofluorescence originated from food matrices (Croote et al., 2019, Lupo et al., 2020), detection techniques with only a single colorimetric or fluorescent signal type were less resistant to interference and often incompetent for rapid analysis of real samples (Ahuja et al., 2014, Lin et al., 2019). For example, pigments in the fruits and vegetables will affect colorimetric analysis (Ahuja et al., 2014), while autofluorescence of some biological samples will interfere with fluorescence measurements (Chen et al., 2020a). Therefore, enzyme-free and label-free biosensors, which are more straightforward to detect OTA and flexibly output colorimetric and fluorescent signal accommodating to specific food matrices, are still highly desired.

As one of the functional nucleic acids nanostructures, DNA G-quadruplex (G4) is a four-stranded self-assembly folded by guanine (G)-rich DNA sequence (Chen et al., 2021, Chen et al., 2021, Mergny and Sen, 2019, Yang et al., 2020a). The most fascinating characteristics of the G4 is that it can associate with hemin (iron (III)-protoporphyrin IX) to form peroxidase mimicking DNAzyme (Du and Dong, 2017, Guo et al., 2017, He et al., 2012, Hoang et al., 2016, Li et al., 2016, Mergny and Sen, 2019, Xi et al., 2020). G4-hemin DNAzyme has been employed as a versatile signal generator in numerous colorimetric, fluorescent, and electrochemical biosensors for rapid sensing a series of targets (Wu et al., 2020, Yang et al., 2020a, Zhang et al., 2020). Despite broad applications, the practical application of the G4-hemin DNAzyme-based colorimetric biosensors has been limited by its relatively low catalytic activity compared with that of horseradish peroxidase (Li et al., 2016). Recently, we have discovered a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at the 3' end of G4, which remarkably boosts the activity of G4-hemin DNAzyme by just adding an adenine nucleotide at the 3' terminals of G-rich sequences (Li et al., 2016). Thus, it is possible to design G4-hemin DNAzyme with excellent catalytic activity for easy signal amplification without any tedious DNA amplification or nanomaterials labeling involved (Xiao et al., 2019).

In this study, DNA G4 assembly was carefully engineered to construct bioenzyme-free, label-free, and environmentally friendly biosensor for rapid and sensitive OTA detection in colorimetric and fluorescent dual modes. First, a new G-rich DNA was designed based on EnEAA (Li et al., 2016) to produce G4-hemin DNAzyme with excellent catalytic activity for colorimetric signal amplification. This signal-amplifying sequence and an adapted anti-OTA aptamer self-assembled into DNA triple-helix switch via Watson-Crick and Hoogsteen base pairings (Chen et al., 2021, Chen et al., 2021, Hu et al., 2017). The OTA-induced G4 of aptamer would open the switch and release the signal-amplifying sequence for colorimetric signal amplification. The G4 structures of AP9 were further utilized to combine with the fluorogenic dye Thioflavin T (ThT) for fluorescent mode. Thus, colorimetric and fluorescent dual modes for rapid and sensitive sensing of OTA were achieved by skillfully engineering DNA G4 assembly for signal amplification without any bioenzymes or nanomaterials involved. At last, the two modes have been successfully applied to detect OTA in samples with intrinsic pigments and autofluorescence, showing their applicability and superiority.