Introduction

Tetracycline (TET) is produced by actinomycetes. It can inhibit the peptide chain extension and bacterial protein synthesis. Because of the effective antimicrobial properties, few side effect and low price, it has been widely used in the treatment of infectious diseases in humans and animals [1]. Nowadays, the abuse of TET has caused serious environmental and health problems [2]. For instance, the antibiotic residues in daily foods such as meat, milk, honey, fish and eggs [3] may lead to the accumulation of TET in human. This will cause damage to the digestive tract and liver, affect the development of the skeleton, and weaken the immune system function [4]. In this concern, China has set the maximum residue limit of TET residues in honey and milk or muscle tissue to 50 μg kg−1, 100 μg kg−1, respectively [5]. Exploring effective sensing approach to facile monitor TET is hence imperative.

Various techniques, including high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis (CE) and enzyme-linked immunosorbent assay (ELISA) etc. have been developed for TET detection [6,7,8,9,10]. By comparison, the recently emerged aptamer-based bioanalytical method attracts extensive attention [11]. The aptamers are 3D structured single-stranded oligonucleotides. They are normally synthesized by Systematic Evolution of Ligands by Exponential enrichment (SELEX) with high affinity and specificity [12]. Compared with protein antibodies and enzymes, the aptamers can be cost-effectively synthesized with high purity and reproducibility. Besides, they also have advantages of small size, high stability, easy to modify, long-term preservation and without immunogenicity [13]. These attributes enable them to be promising in aptamer-based bioanalysis with high affinity and reproducibility [14]. Luo et al. developed CS-AuNPs probe for colorimetric aptasensing of tetracycline [15]. Xu et al. reported a ferrocene/carbon nanofbers-based ratiometric electrochemical aptasensor for detection of tetracycline residues [16]. Despite of the progress, developing new bioassay system for sensitive and specific determination of antibiotics is still a challenge.

Ultrathin two-dimensional (2D) transition metal chalcogenides and transition metal oxides have gained increased attention in the fields of electronics, sensors and catalysis [17]. Particular research interest has focused on integrating these 2D nanosheets into biosensors, stemming from unique structural and electrical properties combing with high loading efficiency for biomolecules [18]. Generally, most transition metal nanosheets have the ability to quench fluorescence, through energy-transfer or electron-transfer from excited fluorophores to nanosheets. And more importantly, the large surface area of 2D nanomaterial can offer more quenching sites, enabling high quenching efficiency and detection sensitivity [19]. Both theoretical calculations and experimental studies have demonstrated that MoS2 nanosheets can physically adsorb aromatic compounds (such as pyridine or purine) and conjugated compounds [20]. Zhu and co-workers have reported high affinity of single-stranded DNA (ssDNA) to MoS2 nanosheets, through Van der Waals interactions between lamellar planes and bases. They also demonstrated that MoS2 nanosheets have a high fluorescence quenching capability towards dye labeled-ssDNA [21]. Herein, a system of VS2 QD/MoS2 nanosheets for use in an aptamer-based fluorometric tetracycline assay has been developed. The TET aptamers were first fabricated on the surfaces of VS2 QDs. Then, the fluorescence of TET aptamer labeled VS2 QDs was quenched by MoS2 nanosheets. When the TET was specifically recognized by the aptamer, the VS2 QDs may be taken away from the MoS2 nanosheets. This will lead to “turn-on” fluorescence of the VS2 QDs. A VS2 QD/MoS2 nanosheets-based fluorescent aptasensing platform for TET is thus constructed.

Experimental section

Chemicals and instrumentation

Specific details of chemicals and instrumentation are provided in the electronic supplementary material. The MoS2 nanosheets were thermolytically obtained following our previously reported method [22]. The VS2 QDs were synthesized via a one-step hydrothermal approach [23]. The concentration of VS2 QDs is ∼2.2 mg mL−1 after purification by silica gel column chromatography. Related details can also be found in the electronic supplementary material.

Fabrication of aptamer-labeled VS2 QDs

The condensation reaction between amino and carboxyl groups [24] is involved to fabricate the TET aptamer labeled VS2 QDs. The detailed procedures can be described as follows: First, 100.0 μL of 500 mM EDC was added into 1.0 mL of VS2 QD suspension (∼2.2 mg mL−1), and the final concentration of QD suspension was 2.0 mg mL−1. Second, the mixture was continuously stirred for 30 min to activate the carboxyl group on the QD surface. Third, 0.9 mL of Tris-HCl was added, the resulting concentration of the carboxyl-activated VS2 QD suspension was 1.0 mg mL−1 (2.0 mL, ~3.7 μM). Finally, incubation of the TET aptamer (3.0 μM) with carboxyl-activated VS2 QDs at room temperature for 15 min, the TET aptamer labeled VS2 QDs were achieved.

Aptamer-based fluorometric tetracycline assay

In a typical process, 2.0 mL of TET aptamer labeled VS2 QDs (1.0 mg mL−1, ~3.7 μM) was first mixed with 30.0 μL of 0.2 mg mL−1 MoS2 nanosheets. Then, 50.0 μL TET solution with tunable concentration was added and mixed thoroughly. After each sample reacted for 25 min under room temperature, the fluorescence emission was measured under the excitation of 360 nm.

Selectivity

The selectivity of the aptamer-based fluorometric TET assay towards several antibiotics (such as OTC, DOX, KAN, OFX, PNC and CIP) was investigated. Briefly, the fluorescence of the VS2 QDs quenched by MoS2 nanosheets (3.0 μg mL−1) was recorded as the initial intensity. After addition of 50 μL (20 μg mL−1) each of the interferential species into the solution (2.0 mL), the fluorescence intensity was recorded again.

TET assay in milk samples

Milk samples were supplied from retail supermarket (Changchun, China) and treated by the following steps. Briefly, 5.0 mL raw milk was mixed with EDTA’s McLinin Protein Buffer (50 μL 400 mM, pH = 5). To deposit protein and dissolve fat and other organic substances in the sample matrix, 20.0 mL trichloroacetic acid was added and mixed for 1 min. The mixture was then centrifuged at 8000 rpm for 20 min to remove the protein, fat and other organic substances. The pH of supernatant was adjusted to 7.5 by 1 M NaOH solution. After filtration, the final solution was used for detection. A certain amount of TET was spiked into the raw milk. The spiked sample was pretreated and analyzed similarly.

Results and discussion

Fluorescence of aptamer-labeled VS2 QDs

The synthesis and characterization of water-soluble monolayer MoS2 QDs have been reported in our previous work [25]. Pronounced blue-shift of excitonic absorption beyond single-layer MoS2 nanosheets and unusual up-conversion photoluminescence at room temperature were unprecedentedly discovered. Following the modified procedures, we prepared VS2 QDs with a relative uniform size of ~3 nm (Fig. S1a and S1b) similarly. Detailed structural characterization, luminescence behavior and promising applications for glutathione and tetracycline determination can be found in recent reports [23, 26]. In this work, another system composed of VS2 QDs and MoS2 nanosheets has been designed for use in aptamer-based fluorometric assay. Specific determination of tetracycline in practical samples has been exemplified.

For analyte determination, the aptamer labeled VS2 QDs was first prepared via the condensation reaction of carboxyl-activated VS2 QDs and amino-terminated TET aptamer. Scheme 1a shows the schematic illustration of the fabrication of the aptamer labeled VS2 QDs. In Fig. 1a, the aptamer labeled VS2 QDs exhibits a similar emission peak at around 448 nm (blue light) with the VS2 QDs under an excitation wavelength of 360 nm. While the fluorescence intensity slightly declines compared to the primary one. When the aptamer labeled VS2 QDs was stored in the dark at 4 °C for 5, 10, 15, 20 days, imperceptible changes in the fluorescence intensity are observed (Fig. 1b). This result indicates that the aptamer labeled VS2 QDs have a good stability in aqueous solution.

Scheme 1
scheme 1

Schematic representation of the preparation of aptamer labeled VS2 QD (a) for aptamer-based fluorometric tetracycline assay (b).

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Fig. 1
figure 1

a Fluorescence emission spectra of VS2 QDs and aptamer labeled VS2 QDs under the same condition. b The photostability of aptamer labeled VS2 QDs during 20 days.

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The MoS2 nanosheets were synthesized via pyrolysis of ammonium molybdate, a layered C3N4 template and sulfur source under a N2 atmosphere [22]. The characteristic ultrathin structure of these nanosheets can be clearly seen from Fig. S2a. Well-resolved crystal lattice with an interplanar spacing of 0.62 nm can be assigned to the distance of (002) crystal plane of hexagonal MoS2. All the diffraction peaks in Fig. S2b can be indexed to the crystalline planes of hexagonal MoS2 (ICDD, reference number, 00–006–0097). The UV-visible absorption spectrum of MoS2 nanosheets reveals a wide absorption in the range of 350 nm to 600 nm. This absorption spectrum is extensively overlapped with the emission spectrum of the aptamer labeled VS2 QDs (Fig. 2a). This behavior implies a possibility of VS2 QD fluorescence quenching by MoS2 nanosheets. Figure 2b compares the fluorescence intensity change of the aptamer labeled VS2 QDs in the absence or presence of MoS2 nanosheets. Extensively weakened fluorescence is observed in the presence of MoS2 nanosheets, since the emitted fluorescence of the VS2 QDs is partially absorbed by the MoS2 nanosheets and the fluorescence is thus quenched.

Fig. 2
figure 2

a UV − vis absorption spectrum of MoS2 nanosheets and fluorescence emission spectrum of aptamer labeled VS2 QDs. b The fluorescence spectra of aptamer labeled VS2 QDs and aptamer labeled VS2 QD/MoS2 nanosheet without and with TET.

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To explore the possible mechanism of fluorescence quenching, the lifetime of the aptamer labeled QD emission was measured and compared with that of the aptamer labeled VS2 QD/MoS2 nanosheets. The fluorescence lifetime of the composite system decreases (Fig. 3), indicating that the fluorescence decay cannot be ascribed to static quenching and inner filter effect [27]. Although the estimated distance between acceptor and donor is less than 10 nm (detailed calculation can be found in the Electronic Supporting Material), the possibility of fluorescence quenching by photoinduced electron transfer still cannot be excluded. More efforts are required in near future to explore the exact mechanism.

Fig. 3
figure 3

The lifetime change of the aptamer labeled VS2 QDs and aptamer labeled VS2 QD/MoS2 nanosheet.

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Aptamer-based fluorometric TET assay

The change in fluorescence intensity of the aptamer labeled VS2 QD/MoS2 system in the presence of TET is studied. Figure 2b depicts that remarkable fluorescence recovery is realized in the presence of 600 ng mL−1 TET. The specific recognition between TET and the aptamer leads to the VS2 QDs detached from the surface of MoS2 nanosheets. The fluorescence is therefore recovered. By monitoring the turn-on fluorescence intensity, the TET may thus be detected.

The mechanism of the aptamer labeled VS2 QD/MoS2 nanosheets for TET detection is schematically represented in Scheme 1b. In the scheme, the interactions between all the species are also indicated. The VS2 QD probe emits intense blue light at 448 nm under UV light excitation of 360 nm. The MoS2 nanosheet serves as fluorescence quenching species. The TET aptamer labeled VS2 QDs are adsorbed on the surfaces of MoS2 nanosheets through van der walls force, the fluorescence under 360 nm excitation is efficiently quenched. When the molecular recognition between TET and the aptamer labeled VS2 QDs takes place, well-folded TET-aptamer complex is formed. The conformation change of the complex decreases the exposure of nucleobases, the VS2 QDs are detached from the surface of MoS2 nanosheets. The fluorescence of the VS2 QD probe is thus restored.

Optimization of assay conditions

The following parameters are optimized: (a) concentration of MoS2; (b) incubation time; (c) tetracycline aptamer concentration; (d) sample pH value. Respective text and Figures on optimization are shown in Fig. S3 in the Electronic Supporting Material. In short, the following experimental conditions are found to give best results: (a) optimal concentration of MoS2: 3 μg mL−1; (b) optimal incubation time: 25 min; (c) optimal tetracycline aptamer concentration: 3 μM; (d) best sample pH value: 7.5.

TET aptasensing performances

Under optimal conditions, fluorescence detection of TET was carried out. Figure 4a shows the tunable fluorescence spectra towards different concentration of TET. The small inset magnified the fluorescence emission behavior at low TET concentration. The plot of TET concentration-dependent fluorescence intensity is presented in Fig. 4b. A good linear relationship between the fluorescence intensity and TET concentration in the range from 1 to 250 ng mL−1 can be observed in Fig. 4c. The linear regression equation is FL = 1.67 × CTET (ng mL−1) + 1007.22. The correlation coefficient is 0.9982. A detection limit of 0.06 ng mL−1 is achieved (3δ/S, δ is the standard deviation of the blank signal and S is the slope of the linear calibration plot). Compared with most of the aptasensing approaches for the detection of TET, the VS2 QD/MoS2 aptamer-based fluorescent assay exhibits a relatively low detection limit (Table 1), showing a promise in food quality and safety control.

Fig. 4
figure 4

a The change of fluorescence spectra with TET concentration. b The trend of fluorescence recovery with TET concentration. c The linear relationship between emission intensity and TET concentration. d The specificity of VS2 QD-based aptasensor towards a series of structurally similar tetracycline derivatives.

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Table 1 TET assay performances compared with other fluorescent sensing approaches
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Selectivity study

A series of derivatives with similar structure to tetracycline (such as OTC, DOX, KAN, OFX, PNC and CIP) are selected to evaluate the possible interference effect on the determination of TET. Under above conditions, 50 μL of 20 g mL−1 TET and each of the antibiotics were added, and the fluorescence intensity was recorded. All data are obtained based on three repetitive measurements. As shown in Fig. 4d, none of these antibiotics causes obvious fluorescence increase. Above study demonstrates a high specificity to tetracycline over other antibiotics, since the aptamer can distinguish even minor structural difference between the target and its analog [31].

Detection of TET in milk samples

To further demonstrate the possibility of the aptasensor for practical application, the concentrations of TET in several milk samples were detected. Since the tetracycline veterinary drugs in all the milk samples are found below the detection limit, a series of spiked samples were prepared by addition of 50 μL different concentration of TET. For each concentration of TET (5 ng mL−1, 20 ng mL−1, 50 ng mL−1 and 100 ng mL−1), three replicate samples were prepared. Under the optimal conditions, the TET concentration of each spiked sample was detected. The relative standard deviation and spiked recovery are presented in Table 2. The recovery of TET is in the range of 96.7–107.1%. The relative standard deviation (RSD) is less than 3.22%.

Table 2 The results of TET detection in milk samples
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Conclusions

Using MoS2 nanosheets as an efficient quencher to the blue fluorescence of VS2 QDs, a VS2 QD/MoS2 nanosheets-based aptasensor for TET determination was developed. The specific recognition between aptamer and target resulted in an increase of the distance between the VS2 QDs and MoS2 nanosheets. The VS2 QDs were thus detached from the MoS2 nanosheets, and the fluorescence was recovered. This aptamer-based fluorescent assay approach has advantages of high sensitivity, low detection limit and high specificity. Its feasibility of determination of TET in milk samples has been demonstrated. Note that, the interference caused by biomatter is a disadvantage for the method working in the UV (excitation at 360 nm). Many samples display strong background UV absorption and fluorescence. In this regards, the UV light used for fluorescence excitation could be screened off by UV absorbers, which may weaken the signal. Thus, there still exists a limitation of the present approach in practical application.