Bi-color FRET from two nano-donors to a single nano-acceptor: A universal aptasensing platform for simultaneous determination of dual targets

https://doi.org/10.1016/j.cej.2020.126017Get rights and content

Highlights

  • MoS2 nanosheet was found to serve as a single acceptor paired with two nano-donors.

  • A simple-to-use aptasensor was developed based on the bi-color FRET using nano-pairs.

  • Simultaneous determination of dual targets can be achieved in a single measurement.

  • This aptasensor exhibited wide linear ranges and low LODs for AFB1 and OTA detection.

  • This work offers a universal aptasensing strategy in advancing multiplexed analysis.

Abstract

Emerging nano-donor and nano-receptor FRET pairs are more advantageous to broad biosensing applications. However, those reported FRET system generally containing a single pair cannot afford multiplexed analysis. Herein, bi-color FRET from two nano-donors to a single nano-acceptor has been adopted to fabricate a simple-to-use aptasensor for simultaneous determination of dual targets in a single run. Aflatoxin B1 (AFB1) and ochratoxin A (OTA) were selected as the analytes because they have synergistic effect to induce enhanced toxicity. Specially, carbon dots (CDs) and CdZnTe quantum dots (QDs), with unambiguous separation of emission peaks, were used as nano-donors to label the aptamers specific for AFB1 and OTA, respectively. These single-stranded aptamers with labels could be stably adsorbed on a single nano-acceptor MoS2 nanosheets surface due to van der Waals force, leading to the occurrence of FRET. When the aptasensor incubated with AFB1 and OTA, the specific binding between aptamers and targets resulted in partial release of the nano-donor labeled aptamers which disturb the FRET process and ultimately led to the fluorescence recovery to some extent. This aptasensor exhibited a wide detection range of 0.01–10 ng mL−1 for AFB1 and 0.02–5 ng mL−1 for OTA with low detection limits (3.3 pg mL−1 for AFB1 and 7.1 pg mL−1 for OTA). This aptasensor provides a new avenue for simultaneous determination of dual mycotoxins due to its advantages of simple operation, good selectivity and high sensitivity. By simply changing the specific aptamer, our strategy will certainly provide a universal aptasensing platform in advancing multiplexed analysis.

Introduction

Fluorescence resonance energy transfer (FRET) presents as a very promising tool for investigation of molecular interactions and molecular structure [1], [2], [3]. Two essential requirements for FRET occurrence are 1) a good spectral overlap for acceptor absorption and donor emission and 2) a close distance (<10 nm) between acceptor and donor [1]. Fortunately, separate donors and acceptors can be brought in close proximity through binding events such as protein interaction, antibody-based immunoreaction, and DNA hybridization [2], [4]. Disturbing the molecular interactions by the specific targets will lead to spatial separation of donor and acceptor probes which make donor–acceptor pair cannot engage in FRET process. As a result, FRET strategy becomes particular prevailing in biosensing owing to its target-specific signal and homogeneous assays without labor-intensive washing and separation steps [4]. In these designs, FRET assays can significantly improve the duration and simplicity of the experiment, in particular, can effectively avoid “false positive” results. Organic fluorophores are commonly served as either energy donor or energy acceptor in traditional FRET systems, for instance, Rhodamine B and methyl-red [5], Pyrene excimer-SYBR Green I [6], and Cy3-Cy5 [7] pairs. However, the optical limitations of organic fluorophores such as photobleaching, low resistance to chemicals, as well as short fluorescent lifetime, have seriously limited their broad biosensing applications [4].

Employing nanomaterials with appropriate optical features, to replace the traditional donor–acceptor pair, is certainly a central strategy to address the limitations of organic fluorophores. As a typical example, CdTe quantum dots (QDs)-Au nanoparticles (NPs), adopted as nano-donor and nano-acceptor pair, has aided advances in assembling biosensors for various targets ranged from small molecules [8], [9] to proteins [10], [11]. In most recent years, a cascade of FRET assays has been designed based on different nano-donor and nano-acceptor pairs like graphene QDs (GQDs)-AuNPs [12], GQDs-carbon nanotubes [13], carbon dots (CDs)-Au nanoclusters [14], [15], CDs-MnO2 nanostructures [16], GQDs-porphyrinic metal-organic frameworks [17], Au nanoclusters-MnO2 nanosheets [18], upconversion nanoparticles respectively paired to Ti3C2 nanosheets [19], Au nanorods [20], AuNPs [21] or ZnS NPs [22]. However, all these systems based on a single FRET pair are generally suitable for a single target detection in one measurement. Apparently, bi-color or multi-color FRET biosensors with an unambiguous separation of different nano-donor’s emission, if available, might provide a forceful technical support in advancing multiplexed analysis in a single measurement.

Mycotoxins, a group of toxic secondary metabolites generated by filamentous fungi, possess lethal threats to human or animals when they are appeared in food/feed [23], [24]. Aflatoxin B1 (AFB1), possessing the highest toxicity among various mycotoxins, has been defined as a group I carcinogen by the International Agency for Research on Cancer (IARC) in 1993 [25]. Ochratoxin A (OTA), listed as group IIB carcinogen by IARC, is another toxic mycotoxin which can be found in various foodstuffs [25]. What makes it worse is that the co-existence of AFB1 and OTA in a single sample leading to an enhanced toxicity owing to synergistic effect [26], [27]. Chromatographic methodologies including high-performance liquid chromatography (HPLC) [28] and HPLC or liquid chromatography coupled with tandem mass spectrometry [29], [30] are primarily used for multi-mycotoxin detection. However, these chromatographic methods suffer from expensive instruments, well-trained professionals and tedious procedures, which limit their applications in practice [31]. Thus, developing a sensitive, accurate, and rapid method for multi-mycotoxin detection is significant for food safety monitoring.

In this work, a simple-to-use aptasensor based on bi-color FRET from two nano-donors to a single nano-acceptor has been developed for simultaneous detection of AFB1 and OTA. Carbon dots (CDs) and CdZnTe QDs were regarded as two different nano-donors of FRET process, to label the respective aptamer against AFB1 and OTA. In another aspect, MoS2 nanosheets were selected as a single nano-acceptor paired to CDs and CdZnTe QDs. The van der Waals force between single-stranded aptamers and MoS2 nanosheets could bring the donor-acceptor pairs in near field and caused the energy transfer from donors to acceptor occur. By taking advantages of bi-color FRET from two nano-donors to a single nano-acceptor and target-induced fluorescence recovery, AFB1 and OTA can be simultaneously quantified in a single run.

Section snippets

Materials

We obtained AFB1, aflatoxin B2 (AFB2), fumonisin B1 (FB1), and ochratoxin A (OTA) from Sigma-Aldrich (USA). N-Hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 3-mercaptopropionic acid (MPA), tris (hydroxymethyl) aminomethane (Tris), and other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). The aptamers possessing sequences of 5′-NH2-TGG GGT TTT GGT GGC GGG TGG TGT ACG GGC GAG GG-3′ (apt1, against AFB1) and 5′-NH2-GAT CGG

Characterization of MoS2 nanosheets

The as-prepared MoS2 nanosheets appeared to be black green in color (Inset a, Fig. 1A). The UV–vis absorption spectrum for MoS2 nanosheets suspension showed four characteristic absorption peaks, which located at 403, 450, 610, and 670 nm, respectively (Fig. 1A) [32], [36]. Two peaks at 610 and 670 nm can be respectively attributed to B and A excitonic peaks, resulting from the K point of the Brillouin zone in two-dimensional MoS2 nanosheet having large lateral dimensions [37]. The other two

Conclusions

In summary, we have designed a simple-to-use aptasensor for simultaneous detection of two mycotoxins on basis of the bi-color FRET from two nano-donors to a single nano-acceptor. In the detection diagram, two nano-donors (CDs and CdZnTe QDs) were used to label the aptamers specific for AFB1 and OTA, respectively. The single-stranded aptamers with two nano-donors could be flatly “lie” on MoS2 nanosheets surface through van der Waals force, which brought donor–acceptor in close proximity and

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank for the financial support from National Natural Science Foundation of China (Nos. 21976071 and 21675066), Foundation of Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, Qingdao University of Science and Technology (No. SATM201807), Jiangsu University Fund (No. 19JDG025), and Cultivation Fund of Young Key Teacher at Jiangsu University (2015).

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