Fluorescent biosensor for sensitive analysis of oxytetracycline based on an indirectly labelled long-chain aptamer

Abstract

A fluorescent assay for oxytetracycline (OTC) detection was presented based on an indirectly fluorescein-labelled aptamer probe, which was fabricated through the partial hybridization of an OTC long-chain aptamer with a FAM-labelled short-chain ssDNA (S1). Upon combination of the target OTC and its aptamer, S1 with quenched fluorescein was released from the probe to the graphene sheet freely. Subsequently, it was hybridized with the complementary ssDNA (C1) and escaped from the quencher graphene to the solution, resulting in the restoration of fluorescence. Benefiting from the labelling of S1 instead of the OTC aptamer directly, the restoration of fluorescence was independent of the long-chain aptamer, perfectly avoiding the negative effects of the intrinsically existing secondary structure. Together with the high affinity of the aptamer for its target, this assay exhibited excellent sensitivity and selectivity. The linear response for OTC was found to be 0.01–0.2 μM with a limit of quantitation of 0.01 μM. Furthermore, the feasibility of the developed assay in a fresh water system and a milk sample was verified through the recovery experiments using spiked samples. This achievement based on such an indirect labelling method is also expected to lay a foundation to realize effective analysis of small molecule pollutants in the environment, for which the specific aptamers are long-chain nucleotide sequences.

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Introduction

Antibiotics are a class of compounds with antibacterial activity, that is, the ability to kill or inhibit the growth of bacteria.¹ During the past several decades, oxytetracycline (OTC), one of the broad-spectrum antibacterial tetracyclines (TCs), has been widely used in agriculture and animal husbandry as a bacterial infection inhibitor or growth promoter. It is characterized by the exceptional chemotherapeutic efficacy against a wide range of Gram-positive and Gram-negative bacteria based on the inhibition of bacterial protein synthesis.² However, due to its abuse, the residue of OTC in the environment or its accumulation in food has caused a series of negative effects, such as drug resistance in bacteria and allergic reactions in the human body.³ On the basis of its potential menace to public health, the European Union Regulation 37/2010 has stated the maximum residue limit (MRL) of 100 μg kg⁻¹ for OTC in raw milk.⁴ In this regard, it is urgent to develop a facile, practical and effective approach for the detection of OTC in environmental water or contaminated food products with high sensitivity and selectivity. Unfortunately, the previously reported methods, including high performance liquid chromatography (HPLC),^5,6 capillary zone electrophoresis (CZE),⁷ surface-enhanced Raman scattering (SERS),⁸ and amperometric or colorimetric methods,^9–12 suffer from tedious procedures for sample pre-treatment or poor specificity for high structural similarity of TC derivatives. They fail to meet the requirements in the fields of environmental monitoring and food safety.

In recent years, great interest has been focused on the application of aptamers in analytical fields.^13,14 Aptamers, single-stranded DNA (ssDNA) or RNA oligonucleotides, exhibit ultrahigh affinity for their target through the formation of unique secondary or tertiary structures.¹⁵ They are seen as an alternative to antibodies for a number of unique features, for example, chemical synthesis, no limitation to targets, easy modification, thermal stability, and in vitro selection through the systematic evolution of ligands by an exponential enrichment (SELEX) process.^16,17 Moreover, researchers have reported that the specificity of aptamers is superior to antibodies, especially for small molecule targets.^18,19 Since Niazi et al. selected a DNA aptamer which can be used to specifically detect OTC in 2008,²⁰ numerous studies have been reported for the development of an aptamer-based biosensor for OTC analysis,^21,22 including electrochemical, colorimetric, light scattering or microcantilever methods.^23,24 Although they have successfully realized quantitative analysis of OTC, many concomitant limitations still exist. For instance, colorimetric detection using gold nanoparticles is an attractive method due to its simplicity and visibility,^21,25 but the high background signal of the colorimetric system, as well as the instability of metallic nanoparticles in biological complexes or environmental samples, is liable to influence the experimental accuracy. Cantilever array sensors further improve the limit of detection (LOD),²² however, time-consuming immobilization, and laborious operation and washing steps, hinder their on-site application.

In our previous work, we designed a fluorescent assay for OTC analysis based on a direct fluorescein-labelled OTC aptamer and quencher graphene.²⁶ The addition of OTC resulted in the conformational change of the OTC aptamer, which was originally adsorbed on the graphene sheet, leading to its escape from the sheet with fluorescence recovery. The recovered fluorescence intensity was increased with increased OTC concentration, which realized the quantitative analysis of OTC. However, it was premised on tedious ionic regulation experiments, which were used to regulate the interaction between graphene and a long-chain aptamer because of the intrinsic disadvantages of the OTC aptamer. Differing from the aptamers of other substances (like ATP, cysteine, and cocaine^27–29), for which the length was shorter than 40 mer, the aptamer of OTC contains 76 bases. As a kind of long-chain aptamer, the existing secondary structure caused a indistinctive structural transformation after binding OTC, which hindered its effective desorption from the graphene sheet^30,31 and further hindered the fluorescence restoration.

Environmental pollutants, including OTC, are mostly small molecule substances, for which the aptamers are inclined to be long-chain nucleotide sequences. In this respect, an effective approach is expected to be developed for the quantitative detection of such a small molecule on the basis of shielding of the negative effects of the long-chain aptamer. Herein, we design a novel recognition probe for the detection of OTC based on an indirectly fluorescence labelled OTC aptamer, which avoids the negative effects brought by the intrinsic secondary structure of a long-chain sequence. The analysis of OTC is independent of the long-chain aptamer, which is special for most of the environmental small molecule pollutants. It solves a common problem in the interaction between graphene and long-chain aptamers during the sensing process. Combined with the inherent superiorities of aptamers, we suppose that this strategy is expected to lay the foundation to realize effective sensor analysis of small molecule pollutants in the environment, for which the specific aptamers are long-chain nucleotide sequences.

Experimental

Materials

OTC, tetracycline (TET), doxycycline (DOX), and chlortetracycline (CTE) were obtained from Aladdin Reagent Co., Ltd (Shanghai, China). Graphite powder (<300 mesh) was purchased from Beijing Chemical Reagents Company. OTC aptamer (5′-CGTACGGAATTCGCTAGCCGAGTTGAGCCGGGCGCGGTACGGGTACTGGTATGTGTGGGGATCCGAGCTCCACGTG-3′) hybridized with the fluorescein amidite (FAM) labelled short sequence (S1, 5′-AATTCCGTACG-FAM-3′) and the complementary ssDNA to S1 (C1, 5′-CGTACGGAATT-3′), were all provided by Takara Biotechnology Co. (Dalian, China) and purified by high-performance liquid chromatography (HPLC). Phosphate buffer solution (PBS, 20.0 mM, pH 7.4) was prepared by mixing the stock solution of Na₂HPO₄ and NaH₂PO₄. Other reagents of analytical reagent grade were purchased from Tianjin Bodi Chemicals Co., Ltd (Tianjin, China). Ultrapure water obtained from a Millipore water purification system (resistivity > 18.0 MΩ cm⁻¹, Laikie Instrument Co., Ltd, Shanghai, China) was used throughout the experiments. All the reagents were used as received without further treatment. All glassware was thoroughly cleaned with chromic acid and rewashed with the ultrapure water.

Instruments

Fluorescence measurements were performed on a Hitachi F-4500 spectrofluorimeter with an excitation wavelength of 494 nm. Transmission electron microscopy (TEM) images were obtained through high-magnification TEM (FEI Tecnai G2 F30 S-Twin). The conformational changes of the reaction system and the groups of prepared material were characterized by in situ Fourier transform infrared spectroscopy (FT-IR) (Bruker VERTEX 70 FTIR). A polymerase chain reaction (PCR) instrument (Applied Biosystems, Veriti 96 well thermal cycler, Model #: 9902) was used to control the temperature of the system during the OTC detection process. All experiments were carried out at room temperature.

Preparation of graphene

A modified Hummers method was employed to synthesize graphene oxide (GO) from the raw material of graphite powders.³² In short, 1.0 g graphite powder was firstly added into 23.0 mL concentrated H₂SO₄ and stirred for 12 h at room temperature, followed by the gradual addition of 3.0 g KMnO₄ with vigorous stirring at 0 °C. After the mixed solution was sonicated at 0 °C for 24 h, 46.0 mL ultrapure water was transferred into the reaction system slowly. The system was then heated at 98 °C for 12 min with vigorous stirring and diluted with 140.0 mL ultrapure water. The reaction was terminated by the addition of 10.0 mL H₂O₂ (30%), and the colour was observed to change to light yellow. Subsequently, the solution was separated by centrifugation, and washed with 5% HCl and ultrapure water several times. Finally, the GO solid was obtained after the product was dried under a vacuum.

Graphene was synthesized through an environmentally friendly hydrothermal route.³⁰ In brief, 0.5 mg mL⁻¹ GO aqueous solution was transferred into a Teflon-lined autoclave and heated at 180 °C for 6 h. A black homogeneous graphene solution was obtained after autoclave cooling to room temperature.

Determination of OTC

For the quantitative determination of OTC, 6.6 μL of the aptamer-based recognition probe (100 μM) and 60 μL graphene (0.6 mg mL⁻¹) were mixed at room temperature. Then the mixture was incubated with various concentrations of OTC at 95 °C for 1 min and 25 °C for 2 min for 10 cycles using the temperature control system of the PCR instrument. After cooling to room temperature, the solution was incubated with 11 μL of C1 (100 μM) for 30 min. Subsequently, the above solution was diluted with ultrapure water to 1000 μL for fluorescence measurements. The fluorescence intensity was monitored at an excitation wavelength of 494 nm and recorded at an emission wavelength of 520 nm. The slits for excitation and emission were both set at 5 nm. The results were reported as mean values of quadruplicates. Other structurally similar antibiotics such as TET, DOX and CTE were examined under the same procedure for the specificity tests.

Pre-treatment of actual samples for OTC detection

Two types of actual samples (tap water and milk) were chosen to evaluate the feasibility of this assay based on the corresponding pre-treatment. A tap water sample was tested without any pre-treatment. Milk was purchased from a local supermarket and stored at 4 °C. To remove those components in milk which could form chelation complexes with OTC such as protein and fat, 5 mL of the milk were mixed with 5 mL of pH 5.0 McIlvaine buffer (containing 20 mM EDTA (pH 8.0) and 0.5% (v/v) trifluoroacetic acid) to denature the milk proteins. Subsequently, the mixture was defatted and deproteinized by centrifugation at 4 °C for 20 min at 8000 rpm. After adjusting the pH value of the supernatant to 7.0 by dropwise addition of NaOH (0.1 M), the solution was filtered using a 0.2 μm syringe filter and rotary evaporated at 40 °C. The precipitate was resuspended with 20.0 mM PBS (pH 7.4, containing 20.0 mM NaCl, 10.0 mM MgCl₂) to prepare the milk sample for OTC detection.³³

Results and discussion

Detection principle

In this work, we designed a fluorescent sensor for OTC analysis with high sensitivity and selectivity based on a novel aptamer-based recognition probe. The probe consisted of two parts, an OTC long-chain aptamer and FAM-labelled short-chain ssDNA (S1), which was hybridized with the 5′-end of the OTC aptamer (Fig. S1 in the ESI†), realizing the indirect fluorescence label of the of OTC aptamer. Scheme 1 illustrated the detection principle of the assay. The aptamer probe was adsorbed on the graphene sheet via the strong π-stacking interactions between the hexagonal cells of graphene and the ring structure in the nucleobases.^30,34,35 The fluorescence of FAM on the probe was quenched by the graphene sheet due to the fluorescence resonance energy transfer (FRET) proximity. In the presence of OTC, the aptamer preferred to capture the OTC molecule, leading to the release of S1 because of the relatively weaker binding force between the OTC aptamer and S1. As mentioned above, due to the π-stacking interactions between the ssDNA and graphene, the released FAM-labelled S1 was still adsorbed on the surface of the graphene with quenched fluorescence. Upon addition of the short-chain ssDNA (C1), which was complementary with S1, the originally adsorbed S1 was hybridized with C1 based on the principle of complementary base pairing. Due to the weak-interaction between the double-stranded DNA (dsDNA) and graphene sheets,³⁶ the formed dsDNA (S1 hybridized with C1) escaped from the graphene sheet to the solution. The increased distance between FAM and graphene hindered the FRET process, producing a restoration of fluorescence, which facilitated the quantitative detection of OTC. On the contrary, in the absence of OTC, the integrated structure of the aptamer probe remained attached to graphene in spite of the addition of the C1.

Scheme 1 Schematic of the fluorescence assay for OTC detection based on an indirectly labelled OTC aptamer.

Benefiting from the indirect labelling of S1 instead of the OTC aptamer, the restoration of fluorescence was independent of the long-chain aptamer with an intrinsic secondary structure, which impacted the desorption of the aptamer from graphene due to its weak conformational change after binding to its target. That is to say, this strategy successfully kept away from the negative effects brought by the traditional direct aptamer labelling.

Characterization

TEM was employed to characterize the micromorphology of the graphene sheet. As shown in Fig. S2 in the ESI,† the resultant graphene through hydrothermal reduction of graphene oxide, was observed with occasional crinkles, folds, and rolled edges, which are the typical characterizations for graphene.³⁷ The typical FT-IR spectra of graphene oxide and graphene are shown in Fig. S3 in the ESI.† The infrared absorption characteristic peaks of graphene oxide at 3400 cm⁻¹ (O–H), 1733 cm⁻¹ (C=O), 1625 cm⁻¹ (C=C), and 1070 cm⁻¹ (C–O) disappeared or dramatically decreased after the hydrothermal reduction process, indicating that most oxygen-containing groups of graphene oxide were reduced. All the evidence above confirmed the formation of graphene.

Feasibility

This experiment design was based on the unscathed affinity of the aptamer to its target in spite of the partial hybridization, as well as the preferential combination of the aptamer with OTC instead of S1.^38,39 To demonstrate the feasibility of our design, in situ FT-IR spectra were employed to monitor the structural changes of the substance in the system. As shown in Fig. 1, with the addition of S1 to the aptamer solution, the FT-IR spectrum of the mixture was similar to that of the single aptamer, while the spectrum of the mixture of aptamer and OTC showed a relatively bigger difference. It could be concluded that OTC caused a more dramatic conformational change of aptamer, resulting in a decrease of the exposure of chemical groups in the OTC aptamer due to its specific folds. When OTC and S1 existed in the aptamer solution simultaneously, the FT-IR spectrum was similar to that of the mixture of aptamer and OTC, indicating that the aptamer preferred to combine with OTC.

Fig. 1 FT-IR spectra of reaction systems containing different substances.

Optimization

Quenching ability of graphene was characterized by the quenching efficiency of fluorescence in the presence of graphene. As shown in Fig. 2a, the fluorescence intensity of the system gradually decreased with the increase of the graphene concentration, proving the perfect quenching effect of graphene by accepting energy or electron from dyes via FRET proximity. The quenching efficiency reached 91% when the graphene concentration was 0.036 mg mL⁻¹. Moreover, the still quenched fluorescence of the system which was tested after 48 h (in the presence of 0.036 mg mL⁻¹ graphene) verified the stability of the quenching ability of graphene. In addition, different batches of graphene were tested to evaluate the effect of the graphene fabrication process on its fluorescence quenching performance. The results showed that their quenching abilities were almost the same, which basically ensured the reproducibility of this method (Fig. S4 in ESI†).

Fig. 2 (a) Fluorescence spectra of the FAM-labelled aptamer-based recognition probe in the presence of graphene. Experiments were carried out in 20.0 mM PBS (pH 7.4) containing 0.66 μM of the aptamer-based recognition probe and different concentrations of graphene. (b) OTC-induced fluorescence restoration based on S1 of varying lengths. Experiments were carried out in 20.0 mM PBS (pH 7.4) containing 0.66 μM of the aptamer-based recognition probe, 0.036 mg mL⁻¹ graphene, 0.1 μM OTC and 1.1 μM C1.

The relationship between the strategy performance and hybridization length of the aptamer-based recognition probe was investigated. The hybridization length depended on the length of S1, which is complementary with the 5′-end of the OTC aptamer. We used the designed assay to test the restoration of fluorescence based on S1 of varying lengths to evaluate the influence of hybridization length. The sequence details of the tested S1 are listed in Table S1 in the ESI.† As shown in Fig. 2b, for the same concentration of OTC, the most significant restoration efficiency of fluorescence was obtained when the length of S1 was 11 mer. Generally, the denaturation temperature (T_M) of ssDNA was dependent on the sequence length. The shorter the length of S1 was, the lower the T_M, leading to the denaturation of the recognition probe even at room temperature. On the other hand, the binding force between the OTC aptamer and S1 increased with the hybridization length. A strong binding force hindered the release of S1, as well as the combination between the OTC aptamer and OTC. Together with the experimental results, the S1 of 11 bases was chosen for the following experiments.

Determination of OTC

Under the optimized experimental conditions, the detection performance of this strategy for OTC was evaluated. Fig. 3a shows the fluorescence restoration depending on various concentrations of OTC. The fluorescence intensity gradually increased with increasing OTC concentration. It revealed that the FAM-labelled S1, which was originally hybridized with the OTC aptamer, escaped from the graphene sheet to the solution on the basis of the hybridization with C1. Inevitably, many factors were responsible for the incomplete restoration of fluorescence, which had little influence on the detection. For example, graphene sheets hindered the combination between OTC and its aptamer. Fig. 3b shows the relationship between the characteristic fluorescence intensity at an emission wavelength of 520 nm and the added OTC (0.01–0.2 μM). The tendency of fluorescence restoration presented a linear response within 0.01–0.2 μM. By linear fitting of those points, the linear regression equation was FI = 76.4 + 486.8C [OTC] (μM) with a correlation coefficient of 0.980. The limit of quantitation (LOQ) of 0.01 μM was the minimum value tested based on the method. Relative standard deviations (RSDs) of 3.0%, 2.6%, and 2.0% in three repetitive assays of 0.01 μM, 0.05 μM, 0.1 μM of OTC respectively were obtained, confirming the good reproducibility of this strategy. Furthermore, the fluorescence intensity of the system without OTC was also determined. Fig. 3c shows that no obvious fluorescence recovery could be detected regardless of the increase of the C1 concentration. The result verified that OTC played a vital role in the release of S1 from the detection probe and the hybridization of S1 and C1.

Fig. 3 (a) Fluorescence intensity of the reaction system in the presence of OTC. Experiments were carried out in 20.0 mM PBS (pH 7.4) containing 0.66 μM of the aptamer-based recognition probe, 0.036 mg mL⁻¹ graphene, 1.1 μM C1 and different concentrations of OTC. (b) Fluorescence intensity as a function of the OTC concentration (0.01–0.2 μM). The inset shows linear fitting of the lower four points. (c) Fluorescence intensity of the reaction system without OTC in the presence of different concentrations of C1. Experiments were carried out in 20.0 mM PBS (pH 7.4) containing 0.66 μM of the aptamer-based recognition probe and 0.036 mg mL⁻¹ graphene.

Table S2 in the ESI† summarized several recently developed sensor approaches for the detection of OTC. It indicated that the minimum value of the analytical ranges (LOQs) achieved in the present assay was comparable to the majority of the listed methods. Compared to the method in the absence of the short-chain S1,²⁶ this approach realized OTC detection with a lower minimum value of 0.01 μM, and the tedious ionic regulation steps which used to regulate the interaction between graphene and long-chain aptamer had been avoided. The excellent sensitivity was attributed to the shielding of the intrinsic secondary structure of the OTC long-chain aptamer via the indirectly labelled method. Furthermore, this assay was carried out in a homogeneous system, avoiding time-consuming immobilization procedures used in common heterogeneous systems.^22,23,25,40 Accordingly, it could be concluded that the present strategy provided a sensitive and simple approach for the quantitative detection of OTC.

Selectivity and ability of anti-interference

To evaluate the selectivity of this fluorescent strategy, we investigated the signal recovery of possible interferences under the above experimental condition. The chosen interferences all belonged to TCs which were structurally similar to OTC, including CTE, DOX, TET. They all had a naphthalene ring and three acidic/basic groups: tricarbonyl, phenolic β-diketone, and dimethylamine. In a typical experiment, the designed assay was incubated with 5 μM CTE, 5 μM DOX, 5 μM TET and 0.2 μM OTC. As shown in Fig. 4, the characteristic fluorescence intensity at the emission wavelength of 520 nm for OTC was 168.5, while it was 77.9 for CTE, 75.1 for DOX, 73.4 for TET, and 82.9 for a mixture of CTE, DOX and TET based on the fluorescence intensity of the blank sample, which was 69.0. Bearing in mind that the concentration of OTC used in this experiment was 25 times lower than those of CTE, DOX and TET, the results indicated that the interferences were incapable of causing effective fluorescence restoration to influence OTC detection, proving good selectivity of the designed assay. Moreover, the anti-interference ability of the strategy was also investigated. The detection of OTC (0.2 μM) was carried out with these chosen interferences (5 μM CTE, 5 μM DOX, 5 μM TET). As shown in Fig. 4, it was clear that the assay realized the quantitative detection of OTC even though a high concentration of interferences existed in the system, which verified the anti-interference ability of our assay. The excellent selectivity and ability of anti-interference were not only ascribed to the high affinity of the aptamer for its target, but also the protection of its recognizing ability due to such an indirect labelling method. Even if there is an extremely slight difference between the target and interferences, the corresponding aptamer could recognize and capture the target accurately.

Fig. 4 Fluorescence intensity in the presence of 0.2 μM OTC, 5 μM CTE, 5 μM DOX, and 5 μM TET. The interference mixed system contained 0.2 μM OTC, 5 μM CTE, 5 μM DOX, and 5 μM TET. Experiments were carried out in 20.0 mM PBS (pH 7.4) containing 0.66 μM of the aptamer-based recognition probe, 0.036 mg mL⁻¹ graphene and 1.1 μM C1.

Detection of OTC in actual samples

Because the abuse of OTC has caused its accumulation in food and residues in the environment, the assay for OTC detection was applied to tap water and milk samples to evaluate its feasibility in an actual aquatic environment and food. The pre-treated samples were tested and the results are shown in Fig. S5 in the ESI.† Compared to the tested data of the ultrapure water system, which was employed as a blank sample, it indicated no OTC detection in these samples. Furthermore, the interference of other ingredients in these actual samples on OTC determination was investigated by recovery experiment using spiked samples. The analytical results are given in Table 1. The recoveries were acceptable and were calculated to be 94% and 96% in the milk samples, and 97% and 105% in the tap water samples based on the addition of 0.10 μM and 0.20 μM OTC. In general, the developed assay could be primarily applied for the determination of OTC in food and fresh water systems.

Table 1 OTC detection in actual samples and spiked recoveries

Sample	Added (μM)	Detected (μM)	Recovery^a (%)
a The results was obtained in four repetitive assays.
Milk	0.10	0.094 ± 0.0025	94 ± 2.5
	0.20	0.192 ± 0.0046	96 ± 2.3
Tap water	0.10	0.097 ± 0.0026	97 ± 2.6
	0.20	0.21 ± 0.0035	105 ± 1.8

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Conclusions

In summary, a fluorescent assay for OTC detection with high sensitivity and selectivity was successfully developed on the basis of an indirectly fluorescein-labelled OTC aptamer. This strategy avoided the negative effects of the intrinsic secondary structure of the long-chain aptamer brought about by the directly labelled method. In the presence of OTC, flexible fluorescence restoration was measured upon combination of OTC and its aptamer, and the release of the FAM-labelled ssDNA from the quencher graphene. The linear detection range was found to be 0.01–0.2 μM with a LOQ of 0.01 μM. Furthermore, the feasibility of the developed assay in the milk sample and fresh water systems was verified through the recovery experiments using spiked samples. This achievement not only realized the detection of OTC, but also laid a foundation of effective quantitative analysis of small molecule substances in the environment, based on its specific long-chain aptamer using such an indirect labelling method.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21277016), Program for Changjiang Scholars and Innovative Research Team in University (IRT_13R05).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Relevant characterization, spectra and experimental data. See DOI: 10.1039/c5ra04025f

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