Ratio fluorescence detection of tetracycline by a Eu3+/NH2-MIL-53(Al) composite

Tetracycline detection has been a great concern because of its overuse and difficulty in degrading. Here, a detection method with ratio fluorescence was developed by synthesizing Eu3+ doped nanocomposites with NH2-MIL-53(Al) nanosheets. After adding tetracycline, the fluorescence intensity at 616 nm characteristic emission peak of Eu3+ was sensitized by the antenna effect generated from coordinating Eu3+ with tetracycline, but the fluorescence of NH2-MIL-53(Al) at 433 nm was quenched by the fluorescence resonance energy transfer between the Eu3+-tetracycline composition and NH2-MIL-53(Al). Therefore, the efficient detection of tetracycline was achieved based on this change of ratio fluorescence signal. The experimental results show that Eu3+/NH2-MIL-53(Al) has excellent selectivity, a wider linear range and a lower detection limit for detecting tetracycline. This method can afford favorable ideas for developing advanced chemical and biological sensors.


Introduction
Tetracycline (TC) has been used to treat bacterial infections due to its excellent antibacterial properties and good therapeutic effects. [1][2][3] It is difficult to be degraded. Therefore, excessive use may cause it to accumulate in foods such as meat, eggs and milk, in water and soil, which seriously threatens human health. [4][5][6] It is necessary to seek a highly sensitive and selective strategy to realize the detection of TC. At present, relatively mature detection methods include immunoassay, liquid chromatography-mass spectrometry, chemiluminescence, high performance liquid chromatography and so on. 7-10 However, their applications are restricted owing to the methods are oen time-consuming, requiring expensive equipment and complex sample preparation. 11 Recently, novel uorescence detection methods based on the design of highly effective uorescence probes have caught widespread attention because of their obvious advantages, such as simple, sensitive, fast and easy operation with a lower cost. A variety of nanomaterial-based uorescent probes have been reported for detecting TC, including water-soluble quantum dots (QD), 12 metal nanoclusters (M NCs), 13 and Ag nanoparticles (Ag NPs). 14 These sensors usually detect the target object through the change of a single uorescent signal. The accuracy of the detection results from these methods can be affected by some human and environmental factors. 15 In order to effectively overcome these problems and achieve sensitive and accurate detection to a target, some ratiometric uorescent probes have been proposed. For example, Li et al. 16 designed a SiNPs-Eu sensor based on uorescence resonance energy transfer for a ratio-type detection of TC. Recently, a new ratiometric uorescence probe based on carbon dots was further developed for detecting TC. 12,17 Liking metal-organic framework materials (MOFs), twodimensional metal-organic framework nanosheets are coordinately assembled by metals and organic ligands. Compared with MOFs, MOFs nanosheets have many advantages, such as rich active sites, large specic surface area, high aspect ratio, adjustable porous structure and chemical composition, as well as ultra-thin thickness, which are widely used in the elds of catalysis, gas separation and chemical sensing. 18,19 However, most MOFs are currently synthesized in organic solvent systems to ensure their topological structure, which leads to poor water stability and limits the application of water-soluble substances in aqueous systems. 20 The introduction of NH 2 -MIL-53(Al) in water solvent through one-pot hydrothermal synthesis of -NH 2 has the advantages of avoiding the complicated steps involved in post-modication and good water solubility. On the other hand, for the FL detection system, the introduction of -NH 2 can improve the selectivity and sensitivity, thus signicantly expanding the potential application of MOFs in FL analysis. [21][22][23][24] The introduced -NH 2 can react with TCs through hydrogen bond interaction, effectively speeding up the quenching of the uorescence of NH 2 -MIL-53(Al). In addition, the MIL-type MOF composed of Al 3+ and dicarboxylate ligand is stable to water and high temperature. 20,[25][26][27] Therefore, efficient sensors can be developed by using the advantages of the composite materials based on MOFs nanosheets.
In this paper, a novel ratio-type uorescence sensor based on the composite material of MOFs nanosheets (Eu 3+ /NH 2 -MIL-53(Al)) was prepared and used for detecting tetracycline. First, the NH 2 -MIL-53(Al) nanosheets were prepared by hydrothermal method. The Eu 3+ /NH 2 -MIL-53(Al) nanocomposites were then obtained by doping Eu 3+ ions into NH 2 -MIL-53(Al) nanosheets. Once tetracycline was added, Eu 3+ acts as a response unit to coordinate with tetracycline to form Eu-TC complex, which sensitizes the characteristic emission peak of Eu 3+ at 616 nm through the antenna effect. With the successive concentration increase of tetracycline, the characteristic the emission peak of Eu 3+ at 616 nm increase continuously. Due to the overlap between the ultraviolet-visible absorption spectrum of Eu-TC complex and the uorescence emission spectrum of NH 2 -MIL-53(Al), there might be a uorescence resonance energy transfer effect between them, making the uorescence intensity of NH 2 -MIL-53(Al) at 433 nm quenched, but the uorescence intensity of Eu-TC complexes increases successively. Therefore, the highly sensitive detection of tetracycline can be achieved by using these ratio uorescence signal change. The experimental results show that Eu 3+ /NH 2 -MIL-53(Al) has a relatively high selectivity, a wide linear range and a low detection limit for detecting tetracycline. The mechanism diagram of the ratio-type detection of tetracycline by Eu 3+ /NH 2 -MIL-53(Al) nanocomposite is shown in Scheme 1.

Chemicals and instruments
Europium oxide, tetracycline and hydrochloric acid are purchased from Aladdin. Tris(hydroxymethyl)aminomethane was obtained from Shanghai Zhongqin Chemical Reagent. Absolute ethanol was from Tianjin Anlong. All reagents are analytical pure grade. Europium chloride was prepared from europium oxide. The water used in the experiment was ultrapure water (18.2 MU cm). Fluorescence spectrum were recorded by a FluoroMax-4 uorescence spectrophotometer (American FEI Corporation). Other instruments are the PHS-3B pH acidity meter (Shanghai Precision Instrument Science Co., Ltd.), the Nicolet Impact-400 Fourier infrared spectrometer (Shimadzu, Nicolet Corporation), the XRD-6000 X-powder diffractometer (Shanghai Rente Testing Instrument Co., Ltd.), the UV-1102 UV spectrophotometer (Shanghai Tianmei Scientic Instrument Company), the DZF-6020 vacuum drying oven (Shanghai Yiheng Technology Co. Ltd), the BSA224S electronic balance (Beijing Sedorius Scientic Instruments) and the PCD-2000 thermostatic blast dryer (Shanghai Langgan Shiyan Shebei Co. LTD.).

Preparation of NH 2 -MIL-53(Al) nanosheets
NH 2 -MIL-53(Al) nanosheets were prepared as follows. 28 3 mmol AlCl 3 $6H 2 O was dissolved into 15 mL ultrapure water. Then, 3 mmol NH 2 -H 2 BDC was added into the above solution under magnetic stirring. Aer stirring for 30 minutes, dropping 15 mL deionized water solution with 6 mmol urea into the above mixture, and stirring for 30 minutes. Furthermore, the prepared mixture was placed in a 70 mL autoclave and reacted in an oven at 150 C for 5 hours. Aer cooling to the room temperature, the mixture was centrifuged at 8000 rpm and washed several times with ultrapure water. Then, the product was dispersed in 20 mL of DMF and 20 mL of methanol, and respectively stirred at room temperature for 1 day. Finally, the solvent was removed by centrifugation, and the desired product was obtained by drying.

Detection TC by NH 2 -MIL-53(Al) nanosheet
At room temperature, the specic process the uorescence detection of tetracycline (pH ¼ 5, 0.1 M) in Tris-HCl buffer solution was as follows. 2 mL Tris-HCl buffer solution (0.1 M, pH ¼ 5) was added into 100 mL (0.005 mg mL À1 ) NH 2 -MIL-53(Al) aqueous solution. Then, the TC solutions with different concentrations were added into the above solution. Finally, the uorescence intensity of NH 2 -MIL-53(Al) nanosheets was measured and observed at an excitation wavelength of 339 nm.

Eu 3+ /NH 2 -MIL-53(Al) nanocomposite testing TC
The specic process of uorescence detection of tetracycline (pH ¼ 9, 0.1 M) in Tris-HCl buffer solution at room temperature was as follows. 2 mL Tris-HCl buffer solution (0.1 M, pH ¼ 9) was added into 100 mL (0.005 mg mL À1 ) Eu 3+ /NH 2 -MIL-53(Al) aqueous solution. Then, the TC solutions with different concentrations were added into the above solution, and reacting for 1 minute at room temperature. Finally, the change of the uorescence signal of Eu 3+ /NH 2 -MIL-53(Al) nanocomposite was measured at the excitation wavelength of 339 nm.

BET characterization of NH 2 -MIL-53(Al) and Eu 3+ /NH 2 -MIL-53(Al)
In order to further characterize the composite material, we use BET characterization, and the result is shown in Fig. 3.

Condition optimization of the TC detection by NH 2 -MIL-53(Al) nanosheets
Before using composite materials to detect TC, a single material NH 2 -MIL-53(Al) nanosheets was used to perform the uorescent detection on TC. In order to improve the accuracy of the detection results, the pH and response time during the experiment were optimized. The uorescence intensity changed with the change of the pH value within the range of 4-9, and had the most response when pH ¼ 5 (Fig. 5A). So, pH ¼ 5 was selected as the optimal pH value. During the experiment, 20 mL TC (10 À3 M) was added dropwise (Fig. 5B). Once TC was added, the uorescence intensity of the nanosheets decreased rapidly, and did not change apparently with the increase of time. It is obvious that the uorescence intensity of NH 2 -MIL-53(Al) can be barely impacted by the response time.

Fluorescence detection of TC by NH 2 -MIL-53(Al)
TC was detected with the above-mentioned optimal conditions. With the TC concentration increase from 0-120 mM, the uorescence intensity at 433 nm gradually decreases (Fig. 6A). Within the range of 1.5-70 mM TC concentration, the change in   uorescence intensity ((F 0 À F)/F 0 ) shows a good linear relationship with the TC concentration (Fig. 6B). The linear regression equation is (F 0 À F)/F 0 ¼ 0.04409 [TC] + 0.01748, and the calculated minimum detection limit (LOD) is 0.92 mM.

Fluorescence detection of TC by Eu 3+ /NH 2 -MIL-53(Al)
The response time of the uorescent probe Eu 3+ /NH 2 -MIL-53(Al) for detecting tetracycline was studied (Fig. 7). The response time between the uorescent signal of Eu 3+ /NH 2 -MIL-53(Al) composite and TC is very fast, which can be completed within 1 min. The uorescence intensity ratio (I 616 /I 433 ) basically unchanges with the further increase of the response time, indicating that the probe has a better response and stability for detecting TC. Fig. 8A shows the response of TC solutions with different concentrations to the uorescence intensity ratio (I 616 /I 433 ) of Eu 3+ /NH 2 -MIL-53(Al) composite material. With the increase of the TC concentration, the uorescence intensity of NH 2 -MIL-53(Al) at 433 nm is getting smaller and smaller, but the uorescence intensity of Eu 3+ is increasing at 616 nm. Therefore, the Eu 3+ /NH 2 -MIL-53(Al) composite material can be used as a ratiometric uorescent probe for detecting TC. Fig. 7B is the relationship between different TC concentrations and the uorescence intensity ratio (I 616 /I 433 ). When the TC concentration changes from 0.5 to 60 mM, the concentration of TC and I 616 /I 433 shows a good linear relationship. The linear equation is I 616 /I 433 ¼ 0.02919 [TC] + 0.05961 with a correlation coefficient

The selectivity of the TC detection by Eu 3+ /NH 2 -MIL-53(Al)
The effect of some interferences like Co 2+ , Ca 2+ , Mg 2+ , Zn 2+ , K + , ascorbic acid (AA), cysteine (Lys) and glutathione (GSH) to the uorescence intensity ratio (I 616 /I 433 ) of Eu 3+ /NH 2 -MIL-53(Al) composite material was studied (Fig. 9). The results show that the uorescence intensity ratio is obviously changed by adding TC, while other interferences have little effect on the uorescence intensity ratio, indicating that the nanocomposite can achieve TC detection.

Detection of TC in water samples
In order to further evaluate the anti-interference ability of uorescent probes and the feasibility of this dual emission ratio uorescent probe for rapid and ultra-sensitive detection of TC in practical applications, we performed uorescence spectroscopy on actual water samples (tap water) test. As shown in the ESI Table 2, † the uorescence intensity ratio of tap water and deionized water solution gradually increases with the increase of TC concentration. In addition, the ratio of the uorescence intensity of the probes in the two water samples was approximately the same, and no signicant difference was observed. The uorescence intensity ratio has a linear relationship with the TC concentration and a good recovery rate of standard addition is obtained. The results show that the detection system has the same results as the laboratory conditions of deionized water, which can effectively eliminate the interference of coexisting substances, which proves that the probe has excellent selectivity. At the same time, it proved the accuracy and reliability of the probe in measuring TC in environmental samples.

The mechanism verication of TC detection by Eu 3+ / NH 2 -MIL-53(Al)
In order to study the proposed detection mechanism, the characterization of ultraviolet-visible absorption spectrum and uorescence spectrum was carried out. There is a spectral overlap between the ultraviolet-visible absorption spectrum of the complex TC-Eu 3+ /NH 2 -MIL-53(Al) and the uorescence spectrum of NH 2 -MIL-53(Al) (Fig. 10), indicating that there may have a uorescence resonance energy transfer effect between them. As an energy donor, the uorescence intensity of NH 2 -MIL-53(Al) is quenched, while the uorescence intensity of the TC-Eu 3+ /NH 2 -MIL-53(Al) complex as an energy acceptor is enhanced. Thereby, detecting tetracycline can be achieved by changing the ratio of uorescence signals.

Conclusion
In summary, a ratio-based uorescence detection method is proposed for detecting TC by preparing Eu 3+ /NH 2 -MIL-53(Al) nanocomposite. When TC is added, Eu 3+ acts as a response unit to coordinate with tetracycline to form a complex, thereby sensitizing the characteristic emission peak of Eu 3+ at 616 nm through the antenna effect. Due to the spectral overlap between the ultraviolet-visible absorption spectrum of the formed complex and the uorescence spectrum of NH 2 -MIL-53(Al), indicating that there may be a uorescence resonance energy transfer effect between them, so the uorescence intensity of NH 2 -MIL-53(Al) at 433 nm is quenched. With the increase of TC concentration, the uorescence at 433 nm decreases continuously, and the uorescence at 616 nm increases. So, the purpose of detecting tetracycline can be achieved by changing the ratio of uorescence signals. The linear range of this ratio uorescent probe for detecting tetracycline is 0.5-60 mM, the minimum detection limit is 0.16 mM. The method provides a novel idea for detecting tetracycline with excellent selectivity.

Conflicts of interest
There are no conicts to declare.