Elsevier

Talanta

Volume 202, 1 September 2019, Pages 279-284
Talanta

Enzyme-free fluorescent detection of microcystin-LR using hairpin DNA-templated copper nanoclusters as signal indicator

https://doi.org/10.1016/j.talanta.2019.05.013Get rights and content

Highlights

  • A simple, low-cost and enzyme-free fluorescent probe was developed for detection of MC-LR.

  • The hairpin DNA-templated copper nanoclusters were used as fluorescent probe.

  • This strategy takes advantage of recognition of MC-LR and its aptamer as well as fluorescent properties of hpDNA-CuNCs.

  • This method does not require complex DNA sequence design, fluorescent dye label or sophisticated experimental techniques.

Abstract

Microcystin-LR (MC-LR) is a potent hepatotoxin that is often associated with blooms of cyanobacteria. The analysis of trace MC-LR plays important role in environmental and health fields. Herein, we developed a low-cost and enzyme-free detection method of MC-LR by using hairpin DNA-templated copper nanoclusters (hpDNA-CuNCs) as fluorescent probe. The hpDNA-template was designed and fabricated by a MC-LR aptamer loop and a double strand stem, which can specifically recognize target MC-LR with strong affinity. The AT-rich and complementary double strand stem serves as a template for the formation of CuNCs. The formed fluorescent sensing probe of hpDNA-CuNCs exhibits maximum emission wavelength at 575 nm. Upon the addition of target MC-LR into the hpDNA-CuNCs, we observed fluorescence was quenched considerably due to the high affinity between MC-LR and hpDNA aptamer strand loop, which indicated a conformational change of hairpin probe from the stem-loop DNA structure to single-stranded DNA. Then, the change of fluorescence intensity can be used to monitor the concentration of MC-LR from 0.005 to 1200 μg L−1 with a detection limit of 0.003 ng L−1. Compared with the previous reports, this method does not require complex DNA sequence design, fluorescence dye label and sophisticated experimental techniques. Moreover, the target MC-LR in real water samples has been detected.

Graphical abstract

A simple, sensitive, and label-free fluorescent probe for microcystin-LR detection based on hpDNA-templated CuNCs as a fluorescent indicator.

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Introduction

Cyanobacterial blooms are currently one of the most pressing global environmental problems, which threaten to public health and the water environments by releasing of cyanotoxins. When algal apoptosis in water blooms, the most widespread cyanotoxins (MCs) are produced from cyanobacteria in fresh and brackish water and cause a tremendous threat to aquatic ecosystem and human health [1]. MCs, a class of cyclic heptapeptide cyanotoxins with more than 100 structurally similar forms, can cause liver and kidney damage and render tumor promotion by inhibiting protein phosphatases 1 and 2A (PP1 and PP2A) [2,3]. Among them, microcystin-leucine-arginine (MC-LR) is the most frequently encountered and widely studied toxin in the family of MCs [[4], [5], [6]]. Due to strong toxicity and ubiquity, World Health Organization (WHO) provides a provisional guideline value of 1 μg L−1 as maximum allowable MC-LR concentration in drinking water in 1998 [7]. Therefore, the development of a simple, sensitive, rapid and robust analytical methods is generally a need for trace monitoring of any analyte, not excluding MC-LR compounds, for which identification is urgently necessary in matter of environmental safety and human health fields.

A variety of analytical methods have been developed to detect MC-LR including high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) [8,9], protein phosphatase inhibition assay (PPIA) [10,11], enzyme-linked immunosorbent assay (ELISA) [[12], [13], [14]], and chemical sensors and biosensors [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]]. Although these methods are precise and sensitive, they require large and cumbersome instruments, well-trained personnel and time-consuming analytical processes. Therefore, it is a challenge to develop a simple, sensitive, and specific detection method of MC-LR. One of the promising alternative method for MC-LR screening is the fluorescent based assay for the specific and efficient recognition between antigen and antibody [3,19,20]. The highly-sensitive fluorescent MC-LR sensors can unambiguously detect MC-LR [[26], [27], [28], [29]].

Recently, ultrasmall fluorescent metal nanoclusters (MNCs) have been used for biochemical analysis due to their remarkable fluorescent properties, excellent biocompatibility, and facile surface modification [30,31]. In addition, fluorescent MNCs have been developed as a new class of fluorophores due to their attractive features and molecule-like properties [[32], [33], [34], [35]]. Among fluorescent MNCs, DNA-templated CuNCs are of particular interest in analytical chemistry owing to its high specificity and easy operation without rigorously controlled temperature, which have been used for development of biosensors [[30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45]]. Su et al. established a fluorescent method to detect the activity of exonuclease III by using hpDNA-templated copper nanoparticles as a fluorescent probe [46]. Fluorescent CuNCs with maximum emission wavelength of 575 nm are first formed by using double-strand DNA (dsDNA) as a template. Upon the addition of exonuclease III, the dsDNA template was digested from 3′ to 5′, and the formation of fluorescent CuNCs was inhibited. Then the activity of exonuclease was monitored by decrease of the fluorescence intensity of dsDNA–Cu NPs. Subsequently, the same strategy was used to develop a fluorescent probe for trypsin and its inhibitor based on DNA hosted Cu nanoclusters [47]. The dsDNA-CuNCs was activated with fluorescence enhancement by adding protamine. They observed the fluorescence was quenched by trypsin. Meanwhile, a fluorescent assay for T4 polynucleotide kinase/phosphatase activity and its inhibitors was designed on the basis of nicking enzyme-assisted signal amplification, and poly(thymine)-templated fluorescent copper nanoparticles was used as a fluorescent indicator [48,49]. Furthermore, hairpin DNA loop was able to increase the fluorescence of DNA-CuNCs for detection of S1 nuclease [50]. Although significant progress on the fluorescent MNCs based detection methods have been achieved, in order to fully realize the capacity of highly sensitive and selective, inexpensive, portable and easy-to-use detection remain challenging because of the limitation of current methods.

In this study, we designed a novel and label-free fluorescent probe for microcystin-LR detection by using hpDNA-templated copper nanoclusters (hpDNA-CuNCs). In this approach, hpDNA was designed by a MC-LR aptamer loop and a double strand stem. The MC-LR aptamer loop acts as specific regions to recognize target MC-LR with strong affinity. The AT-rich and complementary double strand stem serves as an effective template for CuNCs formation. When a target is introduced, the specific combination of the target with the recognition aptamer loop releases the CuNCs nucleation sequence, resulting in the decrease of fluorescent intensity of hpDNA-CuNCs system. Hence, MC-LR could be detected through the changes of the fluorescence intensity of hpDNA-CuNCs. Compared with the previous reported works, this strategy does not require any complex DNA sequence design or fluorescence dye label.

Section snippets

Reagent and apparatus

All the oligonucleotides used in this study were synthesized by Takara Biotechnology Co., Ltd. (Dalian, China) and purified with HPLC. The sequences of these oligonucleotides are listed in Table 1. The 3-(N-morpholino)-propanesulfonic acid (MOPS) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Ascorbic acid (AA) was obtained from Sinopharm Chemical Regent Co., Ltd. (Shanghai, China). Copper sulfate pentahydrate (CuSO4⋅5H2O) was provided from Jinhua Chemical Regent Co., Ltd.

hpDNA-CuNCs sensing strategy

The schematic diagram of the designed label-free fluorescent probe for detection of MC-LR is shown in Scheme 1. The hpDNA-template consists of a MC-LR aptamer loop and a double strand stem. The MC-LR aptamer loop can specifically recognize target MC-LR with strong affinity. The AT-rich and complementary double strand stem serves as a template for CuNCs formation. And the random hairpin loop can facilitate the formation of CuNCs on the hpDNA double strand stem [49]. The hpDNA-template was

Conclusions

In summary, we developed a simple, sensitive, low-cost, and label-free fluorescent probe to detect MC-LR. This strategy takes advantage of the specific recognition of MC-LR and its aptamer strand as well as the excellent fluorescence properties of hpDNA-CuNCs. The fluorescence intensity of hpDNA-CuNCs system decreased upon the addition of target MC-LR due to the high affinity between MC-LR and its aptamer. Then, MC-LR can be quantitatively analyzed through the changes of the fluorescence

Acknowledgements

The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (21565031 and 21665027), YMU-DEAKIN International Associated Laboratory on Functional Materials and Mentor and Sailing Plan of School of Chemistry and Environment, Yunnan Minzu University, PR China.

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