Amino-Functionalized Al-MOF for Fluorescent Detection of

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Amino-Functionalized Al-MOF for Fluorescent Detection of Tetracyclines in Milk Chunhua Li, Li Zhu, Weixia Yang, Xie He, Sheliang Zhao, Xiaoshuo Zhang, Wenzhi Tang, Jianlong Wang, Tianli Yue, and Zhonghong Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06253 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Journal of Agricultural and Food Chemistry

Amino-Functionalized Al-MOF for Fluorescent Detection of Tetracyclines in Milk Chunhua Li a, Li Zhu a, Weixia Yang a, Xie He a, Sheliang Zhao a, Xiaoshuo Zhang, Wenzhi Tang a, Jianlong Wang a, Tianli Yue a, b, c, Zhonghong Li a, b, c* a

College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi

712100, PR China b Laboratory

of Quality & Safety Risk Assessment for Agro-products（YangLing），Ministry of

Agriculture，Yangling, Shaanxi 712100,China c National

Engineering Research Center of Agriculture Integration Test(Yangling), Yangling,

Shaanxi 712100,China

*Corresponding

author. Tel: +86 29 8703 8857; E-mail: [email protected]; [email protected] .

ACS Paragon Plus Environment


ABSTRACT: A fluorescent method for detection of tetracyclines (TCs) in milk was developed by using the NH2-MIL-53(Al) nanosensor synthesized via a one-pot hydrothermal method. The nanosensor had a crystalline nanoplates structure with rich groups of -NH2 and -COOH. The -NH2/-COOH of NH2-MIL-53(Al) reacted with the -CO-/-OH of TCs to form a complex. The electron of -NH2/-COOH from the NH2-BDC ligand transferred to the -CO-/-OH of TCs. -NH2 of the NH2-MIL-53(Al) interacted with the -CO-/-OH of TCs by hydrogen bonding. Quenching efficiency of inner filter effect (IFE) was calculated to contribute 57-89%. The synergistic effect of photoinduced electron transfer (PET) and IFE account for fluorescence quenching. TCs were quantitatively detected in milk samples with recoveries of 85.15 ~ 112.13%, the results were in great accordance with high performance liquid chromatography (HPLC) (P > 0.05), confirming the NH2-MIL-53(Al) nanosensor has potential applicability for the detection of TCs in food matrix. KEYWORDS: Amino-functionalized organic Metal Framework; Nanosensor; Fluorescence detection; inner filter effect (IFE);Tetracyclines; Milk


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 INTRODUCTION Tetracyclines

(TCs),

a

subclass

of

polyketides

with

octahydrotetracene-2-carboxamide skeleton, has been extensively used as broad spectrum antibiotics in therapy of animal infections or feed additive to promote the growth of livestock.1,2 The abuse of TCs has led to antibiotic residues in diary food products, such as meat, fish, milk,3 and honey.4 Excessive residual or continually long-term intake with small-doses of TCs can result in undesirable effects of anaphylactic reaction, gastrointestinal disturbance, hepatotoxicity and promote bacterial resistance to antibiotics. 5-8 Therefore, both the European Union (EU) and the U.S. Food and Drug Administration (FDA) have established the maximum residue limit (MRL) of TCs in milk as 225 nM (100 ng/mL) and 676 nM (300 ng/mL) respectively.

6

Various strategies for detection of TCs in food products have been

widely developed, including high-performance liquid chromatography (HPLC),

9

liquid chromatographyemass spectrometry (LC-MS), 10 capillary electrophoresis (CE), 11

enzyme-linked immunoassay (ELISA).

12

Most of those methods are

time-consuming, expensive, sophisticate and skilled personnel needed, and these disadvantages limited their applications in routine analysis. Therefore, it is indispensable to establish a simple, efficient and sensitive method for the detection of TCs. Fluorescent analysis method is considered as a simple, sensitive, easy-to-operate and low-cost method, which uses fluorescent materials (organic fluorescent dyes or fluorescent nanomaterials) as signal platform. Since fluorescent nanomaterials such as



quantum dots (QDs),

13

carbon dots (CDs),

14-15

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metallic nanoclusters (MNC)

8

and

fluorescent metal-organic frameworks (FMOFs) could be effectively quenched by TCs, fluorescence (FL) quenching based methods have been widely explored. However, their widespread applications are susceptible to more or less inherent drawbacks. For instance, conventional QDs are potential threats to food safety for the presence of heavy metals. In addition, MNC are easily aggregate because of their poor stability, and CDs are too small to separate and purify. Among these fluorescent materials, FMOFs are widely explored owing to their intriguing spectral characteristics as well as infinite tenability, excellent thermal stability, availability of in-pore functionality and outer-surface modification.

16

As chemo-sensors, hitherto

FMOFs have been intensively developed to detect various targets, e.g. metal ions, small molecules,

18

gases,

19

biomarkers,

20

pH values,

21

and temperatures.

22

17

The

fluorescent properties of FMOFs generally have a strong response to their crystalline structures, coordination environment of metal centers, and their interactions with guest species (for example, coordination and hydrogen bonds, π-π interactions), which provide solid rationale for fluorescent sensing. 23 Current exploited FMOFs are mostly synthesized in organic solvent systems to ensure their topological structures, which results in poor water stability and limit applications of the water-soluble matter detection in aqueous systems. Alternatively, incorporation of functional groups (-NH2) on the organic linkers can improve water stability of FMOFs, and provide the binding site even electron transfer capability toward analytes. 24 Introduction of -NH2 by one-pot hydrothermal synthesis MOFs has the advantage of avoiding the intricate


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steps involved in postmodification.

25

On the other hand, for FL detection systems,

-NH2 is immobilized in FMOFs clearly improving the selectivity and sensitivity, thus obviously expand the potential applications of FMOFs for FL analysis.

24, 26-28

The

introduced -NH2 can react with TCs by a hydrogen bonding interaction, which result in FL quenching by the transfer of the electron between TC and the chemosensor. 29 In addition, MIL-type MOFs consisting of Al3+ with dicarboxylate ligands are selected as host materials in this study due to their stability toward water and high temperatures.

27, 28, 30-31

Hence, it is expected that the designed amino-functionalized

Al-MOF may be suitable as a novel and effective fluorescent nanosensor for detection of TCs in milk. To the best of our knowledge, the FL assay to detect TCs based on NH2-MIL-53(Al) nanosensor has not been reported yet. Herein, we have developed a facile one-pot hydrothermal approach to prepare NH2-MIL-53(Al) nanosensor for fluorescent detection of TCs in milk. The fluorescent nanosensor is constructed by using Al3+ as metal source and NH2-BDC as organic ligand (Scheme 1). The richness of -NH2, -COOH, and -OH on the MOF endow the nanosensor with high water stability and serve as bonding sites for TCs detection. Hydrogen bonding interaction between -NH2 of NH2-MIL-53(Al) and -OH of TCs response for the specific recognition. The electrons of -NH2/-COOH from the NH2-BDC ligand transfer to the -CO-/-OH of TCs, leading to FL quenching. And the inner filter effect (IFE) of TCs contributes to the quench effect. The synergistic effect allowed the FL nanosensor to selectively recognize TCs from various interferent (ions, amino acid and saccharide). The proposed strategy is feasible to detect TCs in



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milk samples with satisfied accuracy and recoveries, which are verified herein by comparing the testing results of the NH2-MIL-53(Al) nanosensor with HPLC.

 MATERIALS AND METHODS Reagents. Aluminum (III) chloride hexahydrate (AlCl3·6H2O), urea (CH4N2O), N, N-dimethylformamide (DMF) , oxalic acid, acetonitrile (chromatographic grade) and methanol (chromatographic grade) were from Kelong Reagent Co., Ltd. (Chengdu, China). 2-Aminoterephthalic acid (NH2-BDC) was obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Doxycycline (DOX), tetracycline (TET), oxytetracycline (OTC), ampicillin (AMP), streptomycin (STR), cephalosporin (CEP), kanamycin (KAN), chloramphenicol (CHL) were all purchased from Aladdin (Shanghai, China). Aqueous solutions of different metal ions were prepared from NaCl, CaCl2, MgCl2·6H2O, AlCl3·6H2O, FeCl3·6H2O, Zn(NO3)2, MnCl2·4H2O (Kermel Reagent Co., Ltd., Tianjing). Aspartic acid (Asp), cysteine (Cys), tyrosine (Try), lysine (Lys), L-phenylalanine (Phe), serine (Ser), histidine (His), D-fructose, glucose, lactose, sucrose and starch were obtained from Beijing Solabio Technology Co., Ltd. (Beijing, China). Phosphate buffer solutions (PBS) of various pH values were prepared with different ratios of NaH2PO4, Na2HPO4. All of the stock or standard solutions were prepared using ultrapure water. Unless otherwise stated, all the other chemicals were analytical reagent grade and used as received without further purification.

Synthesis of NH2-MIL-53(Al). NH2-MIL-53(Al) was synthesized according to the hydrothermal method with a certain modification.


28

In brief, 0.724 g

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AlCl3·6H2O was dissolved to 15 mL ultrapure water and then 0.544 g NH2-BDC was added under continuous stirring. After 30 min, 0.288 g urea in 5 mL aqueous was dropped into the mixture to sequentially stir for another 30 min. Then the mixture was transferred to a 35 mL Teflon-lined autoclave and maintained at 150oC for 5 h. After being cooled to room temperature naturally, the resulted yellow precipitates were separated by centrifuging and washing with ultrapure water. Subsequently, the products were successively re-dispersed into 20 mL DMF and methanol under dark stirring for 12 h at room temperature. Finally, the as-prepared NH2-MIL-53(Al) was centrifuged and then dried in a vacuum oven at 50oC overnight.

Characterization of NH2-MIL-53(Al). The morphology of the obtained MIL-53(Al)-NH2 was investigated by using a Nova Nano SEM-450 (FEI, USA) field emission scanning electron microscope (FESEM). Powder X-ray diffraction (XRD) spectra were collected on a D8 ADVANCE A25 X-diffractmeter (Bruker, Germany) from 5 to 45° with Cu Kα radiation. Fourier transform infrared (FT-IR) spectra were recorded on a Vetex70 FT-IR Spectroscope from 4000-400 cm-1 using KBr pellets and the background was subtracted with a resolution of 4 cm-1. A Shimadzu UV-vis 2550 Spectrophotometer was used to record the absorption spectra of samples. FL spectra were obtained from 350-550 nm using Perkin-Elmer LS-55 FL Spectrophotometer (Maryland, USA) with the slit widths of excitation and emission were 10 and 20 nm respectively. HPLC measurements were performed on an Agilent 1200LC HPLC (Agilent, USA).

Fluorescence detection of tetracyclines. In a typical experimental



procedure, 100 μL of MIL-53(Al)-NH2 (0.02 mg mL-1) solution was added into quartz fluorescence cuvette (10*10 mm). Thereafter, different concentrations of tetracyclines standard solution (0-100 μM) or several interferences (Na+, K+, Mg2+, Ca2+ Al3+, Zn2+, Fe3+, Asp, Cys, Try, Lys, Phe, His, fructose, glucose, lactose, and sucrose) were added, and then the system was diluted with PBS (pH=8.0, 0.01 M) to 1.5 mL. After incubating at room temperature for 30 s, the FL spectra were measured from 360 nm to 560 nm upon excitation at 330 nm. All samples for FL measurements were performed in triplicate for statistic purpose.

Fluorescence assay of Tetracyclines in milk. Raw milk was purchased from a local pasture. According to the reported method, 6 1% (v/v) trichloroacetic acid and chloroform was firstly added and mixed under vortex for 5 min to remove the proteins, lipids and other organic substances. Secondly, the samples were sonicated for 20 min and centrifuged at 12000 rpm for 10 min, and then the supernatant was filtered through a 0.22 μM membrane to be used directly for HPLC or FL analysis. Chromatographic conditions were as follows: flow rate of 1.0 mL/min; injection volume of 25 μL; column temperature of 40°C, ultraviolet detector wavelength of 350nm; The mobile phase of A (0.01 M oxalic acid and acetonitrile (92+8,v/v)) and B (Methyl alcohol) for gradient elution (Table S1). Besides, a series of samples containing standard TCs solutions of 5, 20, and 40 μM were prepared by standard addition method to evaluate the applicability. The basic statistics were calculated using Minitab 16.0 software (Minitab Inc., USA).

 RESULTS AND DISCUSSION


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Characterization. The morphology of the obtained NH2-MIL-53(Al) was shown in Figure 1A. The as-synthesized NH2-MIL-53(Al) was nanoplates structure with a uniform thickness of ~ 42.72 nm. As shown in Figure 1B, the XRD pattern of the NH2-MIL-53(Al) nanoplates was similar to the simulated pattern,

32, 33

which

indicated that pure phases were obtained and the structure of NH2-MIL-53(Al) contained chains of cornersharing (via μ2-OH group) [AlO4(OH)2] octahedral being connected through NH2-BDC molecules to form a three-dimensional framework possessing one dimensional pores.

34,

35

The peaks at 8.8° and 10.4°of

NH2-MIL-53(Al) were attributed to unreacted NH2-BDC trapped in the structure. 32, 36 The FT-IR spectra (Figure 1C) indicated that the profile of NH2-MIL-53(Al) nanoplates differed from the spectra of NH2-BDC. The characteristic peaks at 3390 and 3505 cm-1 corresponded to the symmetric and asymmetric stretching vibrations of N-H bonding, indicating that -NH2 was well preserved after coordination between NH2-BDC

and

NH2-MIL-53(Al).

Al3+ 24, 28

which

contributed

remarkable

water-solubility

to

The broad absorption bands at 2500-3300 cm-1 related to

-COOH weakened, indicating that NH2-BDC linked into the framework of NH2-MIL-53(Al) nanoplates with fully deprotonated. 28 Two new absorption peaks of Al-O were observed at 1000-1100 cm-1, which further confirmed that the O atoms within

NH2-H2BDC

linked

to

Al3+

and

the

established

framework

of

NH2-MIL-53(Al). An additional absorption band at 1673-1690 cm-1 was assigned to the stretching vibration of the carbonyl group of unreacted NH2-BDC and DMF, indicating that the NH2-MIL-53(Al) was well activated.


35, 36

The XPS was also


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carried out to analyze the structural composition of NH2-MIL-53(Al) nanoplates, which showed four distinct peaks of O 1s, N 1s, C 1s, and Al 2p at 531.17, 399.17, 284.17, and 74.17 eV, respectively (Figure 2D). The binding energies of the Al 2p at 74.97 eV and 75.77 eV were attributed to Al-O and Al-OH, which evidenced AlO4(OH2) in the NH2-MIL53(Al) nanoplates was formed (Figure S1A).

37

The C 1s

high-resolution XPS spectrum can be fitted by three peaks (Figure S1B) at 284.77, 285.87, and 289.07 eV, which corresponded to C-C/C=C, C-N, and C-O, respectively. 38

The O 1s spectrum was divided into two peaks (Figure S1C) at 532.07 and 533.07

eV, attributing to the Al-O and C=O. 39 In the high-resolution XPS spectrum for N 1s (Figure S1D), there were two peaks at 399.37 and 400.07 assigned to PhNH2 and PhN=C, which was attributed to the nitrogen atom of the amino pendant groups on the organic linker of the framework. 40 Therefore, based on the analysis of FTIR and XPS, the NH2-MIL-53(Al) have been successfully synthesized with richness of -COOH and -NH2, which facilitates their detection performance by electron transfer/ H-bonding interactions. The UV-vis and FL spectra of NH2-MIL-53(Al) were shown in Figure 1E. The UV-vis adsorption peak at ~245 nm might be originated from the π-π* transition in C=C groups and the n-π* transition in C=O groups.

41

The characteristic absorption

peak at 330 nm was attributed to the n-π* transition of C=O or C-OH groups of NH2-MIL-53(Al), which corresponded to the optimum FL excitation peak of 330 nm. The FL spectra showed a symmetric and strong FL emission of NH2-MIL-53(Al) appeared at 425 nm with an optimum excitation at 330 nm, corresponding to the


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photo under an ultraviolet lamp (inset b in Figure 1D,), which resulted from electric transitions relating to surface states. The NH2-MIL-53(Al) exhibited a large Stokes shift of 100 nm, which could avoid crosstalk between the excitation and emission signals.

42

The emission peaks at 425 nm of NH2-MIL-53(Al) were a typically

excitation-independent emission when excitation wavelengths changed from 300 to 360 nm (Figure S2), which might reduce the interference of auto-FL during detection. Moreover, the resultant NH2-MIL-53(Al) presented satisfied FL stability for 3 months (Figure S3) at a wide pH range from 4.0 to 9.0 without significant FL intensity loss (P>0.05) (Figure S4). The quantum yield (QY) of NH2-MIL-53(Al) was measured to be 49.17 ± 2.1%. All these observations indicated the good optical performances of the NH2-MIL-53(Al).

Optimization of TCs detection parameters. In order to optimize the analytical performance of the NH2-MIL-53(Al) nanosensor, some experimental parameters such as the concentration of the nanosensor, the pH of the detection system, the incubation time and temperature were investigated in detail. The effect of NH2-MIL-53(Al) concentration was evaluated by quenching efficiency (QE(%)) of TCs. The quenching efficiency (QE(%)) could be described by Eq. (1), QE %  

F0  F   100%

(1)

F0

where F0 and F were the FL intensities of the nanosensor before and after addition of TCs respectively. The QE(%) of TCs were shown in Figure 2A, It can be see that QE(%) dramatically increased with the concentration from 0.004-0.02 mg/mL, and



then leveled to a stable value, which may be attributed to the almost reaction between the TCs and NH2-MIL-53(Al), and the concentration of 0.02 mg/mL was used to further experiments. The effect of media pH on FL intensity of NH2-MIL-53(Al) was shown in Figure 2B. It can be seen that FL of NH2-MIL-53(Al) quickly increased in the pH range of 4.0-6.0 and insignificantly changed in pH 6.0-10.0, indicating a wide detection pH range, and pH 8.0 was employed in this assay for the subsequent experiments. The effect of incubation time on FL intensity of NH2-MIL-53(Al) after inducing TCs presented in Figure 2C, showed that the FL intensity decreased significantly with the incubation time from 0 s to 30 s, and leveled to stable constant values (P > 0.05), which illustrated that the TCs efficiently reacted with NH2-MIL-53(Al). Accordingly, 30 s was adopted as the optimal incubation time. In addition, the effect of incubation temperature was investigated from 25°C to 70°C and the results were shown in Figure 2D. The fluorescence response of NH2-MIL-53(Al) at the practical incubation temperature range of 25°C to 60°C was nearly unchanged, which indicated that incubation temperature had no significant effect on the TCs detection and room temperature was chosen as the reaction temperature for further experiments. Hence, concentration of 0.02 mg/mL, pH of 8.0, incubation time of 30 s and incubation temperature of room temperature were selected for the development of the method.

Fluorescence sensing of TCs. FL titration experiments were used to quantitatively determine TCs. As seen from the spectra (Figure 3A-C), the FL intensities of the NH2-MIL-53(Al) nanosensor decreased with the concentrations of


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TCs, demonstrating the high sensitivity of the nanosensor. The FL quenching could be described by the Stern-Volmer’s Eq. (2), 15 F0  1  K SV Cq F

(2)

where F0 and F were the FL intensities of the nanosensor before and after addition of TCs respectively; Cq was the concentration of TCs, KSV was the quenching constant (M−1), and in this case Ksv were 0.029μM−1 (DOX), 0.046μM−1 (TET), 0.019μM−1 (OTC) by taking the slope of the regression line in Figure 3D-F. Furthermore, the linear ranges for DOX were 0.00 ~ 66.67 μM with a limit of detection (LOD) of 40.36 nM, 0.00 ~ 72.33 μM for TET with LOD of 26.16 nM, 0.00 ~ 86.67 μM for OTC with LOD of 62.05 nM, where the LOD was estimated based on the following Eq. (3), 26

LOD  3S b / K sv

(3)

where Sb is the standard deviation of the blank sample and Ksv is the slope of the calibration curve. The LOD values were lower than the MRLs of 300 ng/mL (676 nM) and 100 ng/mL (225 nM) TCs in milk set by the U.S. FDA and EU.

6

The

comparison of our method with some other TCs detection methods was shown in Table S2. The proposed method showed equaled LOD, relatively wide detection range and rapid response compared with other FL nanosensors. The above analysis results proved that the NH2-MIL-53(Al) could be employed as an acute candidate for the detection of TCs.

Specificity. Some coexisting potentially interfering substances in milk, including coexisting antibiotics (AMP, STR, CEP, KAN and CHL), common ions (Na+, K+,



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Mg2+, Ca2+ Al3+, Zn2+, Fe3+, Mn2+), amino acids (Asp, Cys, Ser, Try, Lys, His and L-Phe,) and saccharides (D-fructose, glucose, lactose, sucrose and starch), might interfere the TCs detection. Hence, the selectivity and specificity of the as-prepared FL nanosensor were investigated. Foreign coexisting substances were measured by employing the NH2-MIL-53(Al) nanosensor. As illustrated in Figure 4, only TCs induced a prominent FL quenching at the concentration of 100 μM, and the quenching efficiencies (F0/F) of NH2-MIL-53(Al) for the TCs follow the order of TET>DOX >OTC, whereas other coexisting substances had negligible effects on the assay. So the NH2-MIL-53(Al) displayed the highest specific recognition ability for TCs.

Mechanism. FL quenching mechanisms of nanosensor by quenchers were commonly related to the fluorescence resonance energy transfer (FRET), inner-filter effect (IFE) and photoinduced electron transfer (PET).

15, 23, 43

The UV-vis showed in

Figure S5 indicated the absorption spectra of TCs solution had a negligible spectral overlap with the emission spectra of NH2-MIL-53(Al), and therefore the FRET mechanism was excluded. However, the strong absorption of TCs effectively suppressed the excitation energy absorption of NH2-MIL-53(Al) at excitation wavelength of 330 nm, which led to low populated emissive state of NH2-MIL-53(Al) and therefore significantly quenched the FL of the nanosensor. The IFE could be estimated according to Eq. (4), 44

Fcorr 2.3dAex 2.3sAem10 gAem   Fobsd 1  10 dAex 1  10- sAem

(4)

Wherein, Fobsd is the measured maximum FL intensity and Fcorr is the corrected FL


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intensity by removing the IFE from Fobsd; Aex and Aem are the absorbance of NH2-MIL-53(Al) with the addition of TCs at 330 nm and 425 nm, respectively; g is the distance between the edge of the excitation beam and the edge of the cuvette (0.40 cm in this case), while s is the thickness of excitation beam (0.10 cm), and d is the width of the cuvette (1.00 cm). The correction factors (CF) of the IFE (Fcorr/Fobsd) at each concentration of TCs were calculated, which were shown in Table S3. The suppressed efficiency (E%) for the totally observed and the corrected FL of TCs were figured out in Figure 5. We found that the suppressed efficiency of IFE reached 57% for DOX, 69% for TET and 89% for OTC, indicating that the coexistence of other quenching mechanisms was involved. The FTIR of the nanosensor after the introduction of TCs (refer to Figure 1C) showed that the absorption peaks of -NH2 at 3390 and 3505 cm-1 were disappeared and the new board absorption peak of O-H were observed at 3440 cm-1, which ascribed to hydrogen bonding interactions formed between -OH of TCs and -NH2 of NH2-MIL-53(Al).

31

The the strong asymmetric

-C=O stretching absorption at 1680 cm-1 disappeared and a new stretching absorption at 1630 cm-1 appeared and C-N stretching absorption at 1337 to 1349 cm-1 illustrated a complex formed between the -NH2/-COOH of NH2-MIL-53(Al) and TCs. In addition, the red shift of UV-vis spectra of the nanosensor after introduction of TCs was shown in Figure S6, which further confirmed that NH2-MIL-53(Al) did react with TCs to form a complex, and the interaction force was so strong to induce the excited electron of -NH2/-COOH on the surface of NH2-MIL-53(Al) transfering to -CO-/-OH of TCs to quenched the FL.

29

The XRD pattern of NH2-MIL-53(Al) had insignificant



changes after introducing TCs (refer to Figure 1B), which illustrated the high crystalline stability of the nanosensor. Therefore, the highly stabilized nanosensor could be efficient and selective to detect TCs based on the turn-off FL nanosensor.

Detection of Tetracyclines in milk samples. To investigate the applicability of the nanosensor in real samples, the NH2-MIL-53(Al) nanosensor was employed to determinate the concentration of TCs in raw milk samples. No TCs in the raw milk samples were measured (Figure S7D), and the results were corresponding to those obtained by the classical method.

45

The standard addition method was

conducted on the milk samples by spiked with different concentrations (5, 20, 40 μM) of TCs. As summarized in Table 1, the satisfactory recoveries of 85.15 ~ 112.13% with low relative standard deviations (RSD, n = 3) of 0.23% ~ 3.12% were obtained. Furthermore, the routine HPLC was used to verify the reliability of the results detected by FL nanosensor, and Figure S7 showed the chromatograms of DOX, TET and OTC with the retention time of 34.63, 14.32 and 12.73 min, respectively. The detected concentrations by HPLC were well consistent with the NH2-MIL-53(Al) nanosensor (P > 0.05), which indicated that the proposed NH2-MIL-53(Al) nanosensor possessed high accuracy and good reliability for detection of TCs in food samples. In summary, an amino-functionalized Al-MOF nanosensor was developed via one step hydrothermal method for fluorescent detection of TCs in milk. The nanosensor showed rapid FL-quenching response and could detect TCs with acceptable selectivity and sensitivity. The -NH2/-COOH of NH2-MIL-53(Al) reacted with the -CO-/-OH of


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TCs to form the complex. Meanwhile, the electron of -NH2/-COOH from the NH2-BDC ligand transfer to the -CO-/-OH of TCs induced the FL quenching. Besides, the IFE contributed to the quench effect. The nanosensor possesses a wide detection pH and temperature range with a rapid response time. NH2-MIL-53(Al) nanosensor could quantitatively detect TCs in raw milk samples with low LOD and satisfactory recoveries, which showed potential advantages in comparison with HPLC. The present work may provide a novel and practical method for determination of TCs in food matrix. 

ASSOCIATED CONTENT

Supporting Information Scheme 1. Schematic illustration of the NH2-MIL-53(Al) System for Sensing TCs. Figure S1. High-resolution XPS spectra of (A) Al 2p, (B) C 1s , (C) O 1s , (D) N 1s of the NH2-MIL-53(Al). Figure S2. FL emission spectra of NH2-MIL-53(Al) at different excitation wavelengths. Figure S3. FL stability of NH2-MIL-53(Al) for 3 months in room temperature. Figure S4. FL intensity of NH2-MIL-53(Al) at different pH. Figure S5. The UV-vis absorption spectra of TCs and FL spectra of NH2-MIL-53(Al). Figure S6. The UV-vis spectra of NH2-MIL-53(Al) after introduction of TET. Figure S7. Representative chromatogram of standard samples containing100 μg mL−1 TET (A), DOX (B) and OTC (C); Chromatogram of milk sample (D).



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Table S1. Gradient etution program for the analysis of TCs. Table S2. Comparison of reported methods for TCs detection. Table S3. IFE of TCs on the fluorescence of NH2-MIL-53(Al). 

AUTHOR INFORMATION

Corresponding Author *Tel: +86 29 8703 8857. Fax: +86 29 8703 8857. E-mail: [email protected]. Funding This work was supported by National Natural Science Foundation of China (31801628) and Shaanxi Social Development Project (2018SF-401). Notes The authors declare no competing financial interest. 

ABBREVIATIONS USED

TCs, Tetracyclines; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatographyemass

spectrometry;

CE,

capillary

electrophoresis;

ELISA,

enzyme-linked immunoassay; QDs, quantum dots; CDs, carbon dots; MNC, metallic nanoclusters; FMOFs, fluorescent metal-organic frameworks; FL, fluorescence; IFE, inner filter effect; DMF, N, N-dimethylformamide; NH2-BDC, 2-Aminoterephthalic acid; DOX, doxycycline; TET, tetracycline; OTC, oxytetracycline; AMP, ampicillin; STR, ctreptomycin; CEP, cephalosporin; KAN, kanamycin; CHL, chloramphenicol; Asp, Aspartic acid; Cys, cysteine; Try, tyrosine; Lys, lysine; Phe, L-phenylalanine; Ser, serine; His, histidine; PBS phosphate buffer solutions; FESEM, field emission scanning electron microscope; XRD, Powder X-ray diffraction; FT-IR, Fourier


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transform infrared spectra; LOD, limit of detection; MRLs, maximum residue limits; FDA, Food and Drug Administration; EU, European Union; CF, correction factor; RSD, relative standard deviations.





REFERENCES

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Figure captions Figure 1. (A) SEM image of NH2-MIL-53(Al); (B) XRD of NH2-MIL-53(Al), NH2-MIL-53(Al)+TCs;

(C)

FTIR

spectra

of

NH2-BDC,

NH2-MIL-53(Al),

NH2-MIL-53(Al)+TCs; (D) XPS spectrum of the NH2-MIL-53(Al); (E) UV–vis absorption (black line), excitation (red line), and emission (blue line) spectra of NH2-MIL-53(Al) (the insets of (a) and (b) are before and after excited with 365 nm UV lamp). Figure 2. (A) Quenching efficiency (QE(%)) of TCs toward NH2-MIL-53(Al) with different concentrations; (B) FL intensity of NH2-MIL-53(Al) at different pH after introducing TCs; (C) Incubation time versus FL intensity; (D) Incubation temperature versus FL intensity. Figure 3. (A-C) FL emission spectra of NH2-MIL-53(Al) upon addition of TCs at different concentrations; (D-F) the plot of the fluorescence quenching factor (F0/F) versus concentrations of TCs. (Inset: relevant linear regions). Figure 4. FL intensity response of NH2-MIL-53(Al) to 100 μmol/L TCs and interfering species (5-fold concentration of TCs, except ions are 50-fold concentration of TCs) in the absence of TCs. Figure 5. suppressed efficiency (E%) of observed and corrected measurements for NH2-MIL-53(Al) after each addition of different concentrations of TCs, E = 1 – F/F0. F0 and F are the steady-state fluorescence intensities of NH2-MIL-53(Al) in the absence and presence of TCs, respectively.



Page 28 of 34

Table 1. Recoveries (mean ± SD) for the detection of TCs in spiked milk samples. Spiked TCs

DOX

OTC

Recoveries RSD

concentration NH2-MIL-53(Al) HPLC

(%)

(%,n=3)

5

5.15±0.45

5.51±0.08

103.09

0.41

20

19.19±0.32

19.26±0.12 95.97

0.19

40

38.07±0.14

39.24±0.06 95.17

0.06

5

4.26±0.38

5.14±0.14

85.15

0.08

20

18.51±0.26

19.19±0.22 92.56

0.03

40

36.64±0.32

39.03±0.09 91.59

0.71

5

5.61±0.29

5.10±0.10

112.13

0.85

20

20.29±0.45

20.14±0.14 101.46

0.07

40

34.50±0.66

39.26±0.16 86.24

0.04

(μM) TET

TCs founded (μM)


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



TOC Graphic


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Amino-Functionalized Al-MOF for Fluorescent Detection of

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