Sensing Properties of NH2-MIL-101 Series for Specific Amino Acids via Turn-On Fluorescence

Metal–organic frameworks (MOFs) have been demonstrated to be desired candidates for sensing definite species owing to their tunable composition, framework structure and functionality. In this work, the NH2-MIL-101 series was utilized for sensing specific amino acids. The results show that cysteine (Cys) can significantly enhance the fluorescence emission of NH2-MIL-101-Fe suspended in water, while NH2-MIL-101-Al exhibits the ability to sense lysine (Lys), arginine (Arg) and histidine (His) in aqueous media via turn-on fluorescence emission. Titration experiments ensure that NH2-MIL-101-Fe and NH2-MIL-101-Al can selectively and quantitatively detect these amino acids. The sensing mechanism was examined and discussed. The results of this study show that the metal centers in MOFs are crucial for sensing specific amino acids.


Introduction
Amino acids (AAs) are important organic compounds with amino and carboxyl groups and also the essential building units of proteins and enzymes [1]. They are indispensable nutrient compositions in living organisms that are crucially involved in almost all life activities. Abnormal physiological amino acid levels in organisms usually lead to various diseases or serious physiological dysfunctions such as cardiovascular diseases, neurological diseases, diabetes, hepatic failure, kidney malfunctioning, Alzheimer's disease and schizophrenia [2][3][4]. Although there are only 20 basic natural AAs, the combinations of these AAs in different ways make up a tremendous amount of proteins with abundant functions and each amino acid plays an individual characteristic role. For example, cysteine (Cys) is the only one containing the sulfhydryl group among the natural amino acids that may contribute to regulating redox homeostasis and maintaining the spatial structure of proteins [5,6]. Whether an excess or deficiency of Cys could cause some heavy diseases such as abnormal hematopoiesis, neurotoxicity, Alzheimer's disease, retarded growth, edema, muscle/fat loss, hair depigmentation, skin lesions, liver damage and so on [6][7][8]. Lysine (Lys) is a kind of important essential amino acid that cannot be manufactured by the body itself and, thus, must be taken in through daily diet. Lys plays crucial roles in varied biological processes and metabolism, such as the Krebs-Henseleit cycle, polyamine synthesis, carnitine production and so forth [9][10][11]. Meanwhile, the amount of Lys is considered a criterion to evaluate the nutritional level of food as well. Arginine (Arg) is the only one with a guanidine group and the most alkaline one among the natural amino acids. It makes a great contribution to regulating hormone levels and maintaining blood pressure and the immune system, and could also be used to treat various physiological diseases by

Results and Discussion
The phase purities of the bulk as-synthesized powder samples of NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al were ensured by measurements of PXRD (powder X-ray diffraction). As illustrated in Figure S1, the PXRD patterns of the as-synthesized NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al are identical to the simulated one generated from the data of single-crystal X-ray diffraction analysis, demonstrating their phase purities. In addition, the fluorescence properties of NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al in the solid state and in aqueous suspension were investigated at room temperature. As shown in Figure S2, it can be found that NH 2  Next, in order to explore the sensing capacity of NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al toward amino acids, their fluorescence emission spectra in the suspension of 20 kinds of different natural amino acids including Ala (L-alanine), Arg (L-arginine), Asn (L-asparagine), Asp (L-aspartic acid), Cys (L-cysteine), Glu (L-glutamic acid), Gln (Lglutamine), Gly (glycine), His (L-histidine), Ile (L-isoleucine), Leu (L-leucine), Lys (Llysine), Met (L-methionine), Phe (L-phenylalanine), Pro (L-proline), Ser (L-serine), Thr (L-threonine), Trp (L-tryptophan), Tyr (L-tyrosine) and Val (L-valine) with a concentration of 0.1 M were recorded in the range of 375-690 and 360-660 nm excited at 356 and 340 nm, respectively. As depicted in Figure 1, it was worth noting that the emission of NH 2 -MIL-101-Fe was tremendously enhanced by Cys (up to 158 times), while significant increments in the emission intensities in the suspension of Lys (about 3.9 times), Arg (about 3.1 times) and His (about 2.5 times) could be observed for NH 2 -MIL-101-Al. could obviously affect the sensing capacity of NH2-MIL-101-Fe toward Cys, indicati their reasonable anti-interference. Moreover, the sensing mechanism of NH2-MIL-101toward Cys and of NH2-MIL-101-Al toward Lys/Arg could be ascribed to the structu collapse, while the sensing capacity of NH2-MIL-101-Al toward His was concerned w the adsorption of His into the voids of NH2-MIL-101-Al.

Results and Discussion
The phase purities of the bulk as-synthesized powder samples of NH2-MIL-101and NH2-MIL-101-Al were ensured by measurements of PXRD (powder X-ray diffr tion). As illustrated in Figure S1, the PXRD patterns of the as-synthesized NH2-MIL-10 Fe and NH2-MIL-101-Al are identical to the simulated one generated from the data of s gle-crystal X-ray diffraction analysis, demonstrating their phase purities. In addition, t fluorescence properties of NH2-MIL-101-Fe and NH2-MIL-101-Al in the solid state and aqueous suspension were investigated at room temperature. As shown in Figure S2          As we know, the anti-interfering capability is one of the most important evaluation criteria for sensing materials. Therefore, we carried out competing experiments through the first addition of other AAs in H 2 O followed by addition of Cys and Lys/Arg/His to the corresponding suspension of NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al to verify their anti-interference. For NH 2 -MIL-101-Fe, the addition of all the other interfering amino acids had no significant effect on the sensing capability toward Cys except Lys, Asp, His and Arg (Figure 3a). It can be observed that the addition of Lys, Asp, His or Arg could also enhance the emission intensity of NH 2 -MIL-101-Fe, which only resulted in a limited increment in the emission intensity upon the successive addition of Cys. Meanwhile, it is clearly indicated in Figure 3b As we know, the anti-interfering capability is one of the most important evaluation criteria for sensing materials. Therefore, we carried out competing experiments through the first addition of other AAs in H2O followed by addition of Cys and Lys/Arg/His to the corresponding suspension of NH2-MIL-101-Fe and NH2-MIL-101-Al to verify their antiinterference. For NH2-MIL-101-Fe, the addition of all the other interfering amino acids had no significant effect on the sensing capability toward Cys except Lys, Asp, His and Arg (Figure 3a). It can be observed that the addition of Lys, Asp, His or Arg could also enhance the emission intensity of NH2-MIL-101-Fe, which only resulted in a limited increment in the emission intensity upon the successive addition of Cys. Meanwhile, it is clearly indicated in Figure 3b  In addition, we also made efforts to investigate the sensing mechanism of NH2-MIL-101-Fe and NH2-MIL-101-Al toward specific amino acids. Generally, the primary concern of the sensing mechanism of MOFs is the structural stability of the sensing materials. Therefore, we employed PXRD to examine the stability of NH2-MIL-101-Fe and NH2-MIL-101-Al in the solution of amino acids. As shown in Figure 4  In addition, we also made efforts to investigate the sensing mechanism of NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al toward specific amino acids. Generally, the primary concern of the sensing mechanism of MOFs is the structural stability of the sensing materials. Therefore, we employed PXRD to examine the stability of preliminarily that the sensing capability of NH 2 -MIL-101-Fe toward Cys and of NH 2 -MIL-101-Al toward Lys and Arg could be attributed to the structural collapse and such mechanisms can be excluded for NH 2 -MIL-101-Al toward His. To further investigate the intrinsic reason, we speculate that it is the encapsulation of His into the pores of NH 2 -MIL-101-Al that causes the fluorescence emission intensity enhancement based on the investigation of our previous work [38]. Thus, we employed 1 H NMR and FT-IR measurements to verify this speculation. After digesting in NaOH/ D 2 O, we measured the 1 H NMR spectra of the untreated and His-treated NH 2 -MIL-101-Al in D 2 O, and the results are depicted in Figure S3. Compared to the 1 H NMR spectra of NH 2 -MIL-101-Al, it could be found that some new peaks belonging to His appeared in that of His-treated NH 2 -MIL-101-Al ( Figure S3a), suggesting the adsorption of His into the pores of NH 2 -MIL-101-Al. In addition, it could be observed that a new C-C stretching vibration band belonging to His around 1142 cm −1 appeared and the O-H stretching bands belonging to solvent water molecules were greatly weakened in the FT-IR spectra of His-treated NH 2 -MIL-101-Al ( Figure S3b), suggesting that the solvent molecules were exchanged by His. Such a mechanism of collapse of the framework or encapsulation of the analyte into the pore of the framework makes the linear sensing response in the mM range (Figure 2), rather than µM. It means that the amount of analyte in the mM range is essential to destroy the framework or to enter the pore of the framework, resulting in enhancement of fluorescence emission, while the fluorescence sensing with electron transfer and/or energy transfer mechanism gives high sensitivity in the µM range [45]. Furthermore, alternative chemical spectroscopy approaches such as surface-enhanced Raman scattering (SERS) spectroscopy can provide very high sensitivity with a range as low as nM or even pM and aM concentrations [46,47]. In short, the sensing sensitivity depends on mechanism as well as chemical spectroscopy. MIL-101-Al in the aqueous solution of Lys/Arg all disappeared, indicating the collapse of the structures of NH2-MIL-101-Fe and NH2-MIL-101-Al. Therefore, it could be concluded preliminarily that the sensing capability of NH2-MIL-101-Fe toward Cys and of NH2-MIL-101-Al toward Lys and Arg could be attributed to the structural collapse and such mechanisms can be excluded for NH2-MIL-101-Al toward His. To further investigate the intrinsic reason, we speculate that it is the encapsulation of His into the pores of NH2-MIL-101-Al that causes the fluorescence emission intensity enhancement based on the investigation of our previous work [38]. Thus, we employed 1 H NMR and FT-IR measurements to verify this speculation. After digesting in NaOH/ D2O, we measured the 1 H NMR spectra of the untreated and His-treated NH2-MIL-101-Al in D2O, and the results are depicted in Figure  S3. Compared to the 1 H NMR spectra of NH2-MIL-101-Al, it could be found that some new peaks belonging to His appeared in that of His-treated NH2-MIL-101-Al ( Figure S3a), suggesting the adsorption of His into the pores of NH2-MIL-101-Al. In addition, it could be observed that a new C-C stretching vibration band belonging to His around 1142 cm −1 appeared and the O-H stretching bands belonging to solvent water molecules were greatly weakened in the FT-IR spectra of His-treated NH2-MIL-101-Al ( Figure S3b), suggesting that the solvent molecules were exchanged by His. Such a mechanism of collapse of the framework or encapsulation of the analyte into the pore of the framework makes the linear sensing response in the mM range (Figure 2), rather than µM. It means that the amount of analyte in the mM range is essential to destroy the framework or to enter the pore of the framework, resulting in enhancement of fluorescence emission, while the fluorescence sensing with electron transfer and/or energy transfer mechanism gives high sensitivity in the µM range [45]. Furthermore, alternative chemical spectroscopy approaches such as surface-enhanced Raman scattering (SERS) spectroscopy can provide very high sensitivity with a range as low as nM or even pM and aM concentrations [46,47]. In short, the sensing sensitivity depends on mechanism as well as chemical spectroscopy.  Finally, another phenomenon also attracted our interest: the fluorescence emission enhancement ratio of Cys for NH2-MIL-101-Fe was so high. In consideration of the oxidizing capacity of the metal centers of NH2-MIL-101-Fe and the reducing ability of Cys together with the structural collapse of NH2-MIL-101-Fe in the solution of Cys, we speculated that it may be attributed to the redox reaction between Cys and the metal centers of Fe 3+ in NH2-MIL-101-Fe. To verify this, 1 H NMR and high-resolution mass spectrometry (HRMS) measurements were carried out. As shown in Figure S4, it could be found that the 1   Finally, another phenomenon also attracted our interest: the fluorescence emission enhancement ratio of Cys for NH 2 -MIL-101-Fe was so high. In consideration of the oxidizing capacity of the metal centers of NH 2 -MIL-101-Fe and the reducing ability of Cys together with the structural collapse of NH 2 -MIL-101-Fe in the solution of Cys, we speculated that it may be attributed to the redox reaction between Cys and the metal centers of Fe 3+ in NH 2 -MIL-101-Fe. To verify this, 1 H NMR and high-resolution mass spectrometry (HRMS) measurements were carried out. As shown in Figure S4, it could be found that the 1 Figure S5). On the other hand, reversibility and recyclability are also not available for NH 2 -MIL-101-Al to detect His, which may be reasoned by strong interactions between His and the framework since it was difficult to desorb His from the pores of NH 2 -MIL-101-Al. Such strong interactions together with the exchange of solvent molecules with His as mentioned above will increase the rigidity and decrease the non-radiative decay [48]; therefore, fluorescence enhancement was observed when His was added into the NH 2 -MIL-101-Al suspension.

Materials and Methods
All chemicals were received from commercial sources and used directly without purification. The sample of NH 2 -MIL-101-Fe was fabricated according to the method reported previously [49] and the synthetic procedure of NH 2 -MIL-101-Al was followed according to the previously reported literature [50]. PXRD measurements were performed on a Bruker D8 X-ray diffractometer with a Cu-Kα radiation source (λ = 1.5418 Å) under 40 kV and 40 mA. FT-IR-ATR spectra within the range of 400-4000 cm -1 were recorded on an infrared spectrophotometer (Bruker Tensor II) with a diamond ATR module. Fluorescence and 1 H NMR spectral data were obtained by using a Perkin Elmer LS-55 fluorescence spectrometer and a Bruker-DRX (500 MHz) NMR instrument, respectively. HRMS data were achieved on a Thermo Scientific Q Exactive electrospray mass spectrometer. The

Conclusions
In this work, we investigated the sensing capability of amino-functionalized MIL-101 with different metal centers, namely NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al, toward natural amino acids. The results of sensing experiments demonstrated that NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al could detect Cys and Lys/Arg/His via a fluorescence turnon effect, respectively. The results of titration experiments show that the fluorescence enhancement has linear relationships with the concentrations of the analytes within a certain concentration range. Meanwhile, they also exhibit reasonable anti-interference except that Lys, Asp, His and Arg could affect the sensing capability of NH 2 -MIL-101-Fe toward Cys. Moreover, the sensing capacity of NH 2 -MIL-101-Fe for Cys and of NH 2 -MIL-101-Al for Lys/Arg could be ascribed to the structural collapse, while the detection mechanism of NH 2 -MIL-101-Al for His could be attributed to the adsorption of His into the pores of NH 2 -MIL-101-Al. Furthermore, the redox reaction between Cys and Fe 3+ of NH 2 -MIL-101-Fe was responsible for the ultrahigh fluorescence enhancement.
Supplementary Materials: The following are available online: Figure S1: PXRD patterns of the as-synthesized NH 2 -MIL-101-Fe and NH 2 -MIL-101-Al and the simulated one; Figure