Regulating Fluorescent Aptamer-Sensing Behavior of Zeolitic

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Biological and Medical Applications of Materials and Interfaces

Regulating Fluorescent Aptamer-sensing Behavior of Zeolitic Imidazolate Framework (ZIF-8) Platform via Lanthanide Ions Doping Ya-Bo Hao, Zhen-Shu Shao, Chen Cheng, Xiaoyu Xie, Jie Zhang, Wen-Jun Song, and Huaisong Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b12253 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 12, 2019

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ACS Applied Materials & Interfaces

Regulating Fluorescent Aptamer-sensing Behavior of Zeolitic Imidazolate Framework (ZIF-8) Platform via Lanthanide Ions Doping Ya-Bo Hao†,‡, ⊥ , Zhen-Shu Shao†, ⊥ , Chen Cheng†, Xiao-Yu Xie § , Jie Zhang‡, Wen-Jun Song‡, Huai-Song Wang*,†

†

Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China

‡ Tianjin

§

Key Laboratory of Food Biotechnology, Tianjin University of Commerce, Tianjin 300134, China

School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an 710061, China

ABSTRACT: Nanoscale metal-organic frameworks (NMOFs) have been proved to be effective quenching platforms for fluorescent detection of DNA via fluorophore-quencher pairs. Zeolitic imidazolate framework-8 (ZIF-8) is one type of the most promising NMOFs because of its excellent biocompatibility and easy preparation. However, ZIF-8 is rarely used as platforms for fluorescent sensing of DNA because of its bad fluorescence quenching property. In this study, lanthanide ions were doped into ZIF-8 to regulate its fluorescence quenching behavior. The La3+ doped ZIF-8 (ZIF-8-La) showed the best quenching efficiency on dye-labeled DNA. The signal-to-background ratio was around 3 times higher than ZIF-8. Furthermore, a core-shell La3+ doped ZIF-8 (CS-ZIF-8-La) was designed to modify more La3+ on the surface of ZIF-8. Compared with ZIF-8-La, the CS-ZIF-8-La exhibited the same fluorescence sensing behavior toward positive-dye labeled DNA, but showed completely contrary quenching property on the negative-dye labeled DNA. Based on this phenomenon, CS-ZIF-8-La was successfully used as quenching platform for designing ratiometric sensor for DNA and microRNA.

Keywords: Metal-organic frameworks; Lanthanide ions; DNA/microRNA; Ratiometric sensing; Fluorescence quenching

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INTRODUCTION Metal-organic frameworks (MOFs) are a class of high-porous hybrid materials built by metal centers and organic ligands. The extremely versatile structure of MOFs has enabled them to be useful in various applications, including gas storage, catalysis, separation, luminescent sensing, and many others.1-4 Recent years, nanoscale MOFs (NMOFs) for biomedical applications (e.g. bioimaging and drug delivery) have attracted considerable attentions.5,6 Especially, many NMOFs, such as MIL-1017, UiO-66-NH28, and MIL-88A9, have been utilized as quenchers for fluorescent sensing of DNA and microRNA. These NMOFs exhibit that their adsorption capacity toward single-stranded deoxyribonucleic acid (ssDNA) is better than double-stranded deoxyribonucleic acid (dsDNA). Compared with conventional fluorescent sensing nano-platforms (e.g. gold nanoparticles and graphene oxide), the NMOFs have simpler preparation processes, and also show ideal fluorescence quenching ability. Therefore, NMOFs could be able to recognize DNA or microRNA biomolecules by the changes of fluorescence intensity. Zeolitic imidazolate framework 8 (ZIF-8), composed of Zn2+ and 2-methyl imidazole (Figure 1a), is one of the most promising NMOFs because of its large surface area and excellent biocompatibility.10-12 The particle size of ZIF-8 can be easily scaled down to the nanoscale level by changing the reaction solvent at room temperature.13 In biomedical applications, the nanoscale ZIF-8 was mainly used as an ideal vehicle with high loading capacity for controlled release of therapeutic agents.14,15 However, it was rarely used as platforms for fluorescent sensing of DNA and microRNA, because of the bad fluorescence quenching property of ZIF-8 (Figure 1b). Herein, in order to regulate the fluorescence quenching behavior of ZIF-8 to obtain novel nanoquenchers, lanthanide ions (Ln = La3+, Ce3+, Nd3+, and Tb3+ respectively) were doped into the ZIF-8. Because we have investigated, in our previous work, that the Ln based NMOFs show effective fluorescence quenching ability toward fluorophore-labeled aptamers based on the charge transfer (CT) from fluorophores to the lanthanum ions.16 The initial ZIF-8 was prepared in methanol at room temperature firstly. And then, the ZIF-8 was dispersed in Ln3+ solution. The Ln3+ can be confined within the pores of ZIF-8, and ion exchange can also be occurred between Zn2+ and Ln3+. We found the Ln3+ doped ZIF-8 nanoparticles (named as ZIF-8-Lns) show better fluorescence quenching ability compared with ZIF-8 during the fluorophore-aptamer based DNA


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sensing. After adding target ssDNA in the system, the generated fluorophore-labeled dsDNA can leave away from the ZIF-8-Ln surface, followed by obviously fluorescence recovery (Figure 1c). Furthermore, the fluorescent DNA sensing procedure might be affected by the position of Ln3+ in ZIF-8, because there are electrostatic interactions between Ln3+ and negatively charged fluorophores. Therefore, another type of Ln3+ doped ZIF-8 was prepared (named as CS-ZIF-8-Ln): the Ln3+ ions were mainly surface modified on ZIF-8. A different fluorescence quenching phenomenon was observed on CS-ZIF-8-Lns. That is, the aptamers labeled with negatively charged fluorophores (such as FAM or Cy5) can be adsorbed on the surface of CS-ZIF-8-Lns; meanwhile, the fluorescence intensity of the fluorophores can be partly quenched in a certain density. After the interaction with target ssDNA, the emission of FAM or Cy5 was further decreased, which is different from that of positively charged fluorophores (such as TAMRA or Texas red) (Figure 1d). This phenomenon was successfully used for designing ratiometric sensors for ssDNA and microRNA.



Figure 1. Schematic illustration of the fluorescent DNA/microRNA sensing on the platform of ZIF-8 or lanthanide ion (Ln3+) doped ZIF-8. (a): Preparation of ZIF-8. (b-d): Target-induced fluorescence change of fluorophore-labeled aptamers which have been initially adsorbed onto the ZIF-8 (b), ZIF-8-Lns (c), and CS-ZIF-8-Lns (d).

EXPERIMENTAL SECTION Preparation of ZIF-8. A solution of 2-methyl imidazole (2.5 mmol, 0.205 g) in MeOH (50 mL) was added into 50 mL MeOH containing 0.274 g (1.25 mmol) ZnAc·2H2O. After stirred for 15 min at 25 °C, the prepared ZIF-8 was centrifuged and washed with methanol and H2O for three times respectively.


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Preparation of ZIF-8-Lns. Typically, the powder of ZIF-8 (200 mg) was dispersed in H2O (10 mL), and then added into a solution of La(NO3)3 [or Ce(NO3)3, Nd(NO3)3, TbCl3 respectively] (0.4 mmol) in H2O (5 mL) under stirring at 25 °C. After 24 h, the prepared ZIF-8-La (or ZIF-8-Ce, ZIF-8-Nd, ZIF-8-Tb) was collected and washed with H2O by centrifugation for three times.

Preparation of CS-ZIF-8-La. The powder of ZIF-8 (16 mg) was dispersed in EtOH (9 mL), and then 2 μL EtOH solution of H2sq (1 mM) was added under stirring. The reaction was performed at 25 °C for 16 h. The H2sq functionalized ZIF-8 (ZIF-8-sq) was centrifugated and washed with EtOH for three times. Then, the prepared ZIF-8-sq was re-dispersed in 10 mL EtOH containing 1 mM La(NO3)3 and 1 mM H2sq. The mixture was stirred for 24 h at 25 °C. The ZIF-8 surface modified with La3+ ions (CS-ZIF-8-La) was prepared.

Fluorescence quenching ability of the functionalized ZIF-8. The fluorescent DCF (0.5 nM) was mixed with ZIF-8-Ln (or ZIF-8-La, ZIF-8-Ce, ZIF-8-Nd, ZIF-8-Tb respectively) (0.4 mg·mL-1) in MeOH. The emission of the mixture was then tested.

Fluorescent DNA sensing by ZIF-8-Lns. A 20 nM dye-labeled P1 (FAM-P1 or TAMRA-P1) solution was added into the suspension of ZIF-8-Ln (~0.2 mg·mL-1) in phosphate buffer solution (PBS) (10 mM, pH 7.4). After incubated for 5 min, the matching DNA T1 chains (1-50 nM) were mixed with the suspension above for another 5 min at 25 °C. Then, the emission spectra were detected.

Fluorescent DNA sensing by CS-ZIF-8-La. The dye-labeled P1 (40 nM of FAM-P1, 40 nM of Cy5-P1, 20 nM of TAMRA-P1 and Texas red-P1 respectively) was added into the CS-ZIF-8-La suspension (~0.2 mg·mL-1) in PBS (10 mM, pH 7.4) for 5 min. Then, the matching DNA T1 chains (1-50 nM) was introduced and incubated for 5 min at 25 °C. The emission spectra were then detected. All the fluorescent measurements were performed under room temperature.

Ratiometric DNA sensing. Two ratiometric probes (RP-1 and RP-2) were prepared as follows:



(1) The RP-1 was prepared by adding ~0.2 mg CS-ZIF-8-La nanoparticles into 1 mL PBS (10 mM, pH 7.4) solution containing 40 nM FAM-P1 and 20 nM TAMRA-P1. The mixture was stirred at 25 °C for 2 h . The as-prepared RP-1 was obtained under 11 000 r.p.m. centrifugation. Then it was re-dispersed in PBS (10 mM, pH 7.4) for further experiments. (2) The RP-2 was prepared by adding ~0.2 mg CS-ZIF-8-La nanoparticles into 1 mL PBS (10 mM, pH 7.4) with 40 nM Cy5-P1 and 20 nM Texas red-P1. Then, the RP-2 was obtained after treated with the same procedure as RP-1. The ratiometric DNA T1 sensing: T1 was added into the RP-1 or RP-2 (~0.2 mg·mL-1) in PBS buffer (10 mM, pH 7.4). The concentration of T1 was ranged from 1 to 30 nM. After incubation for 5 min, the fluorescence of the mixture was measured under λex = 480 nm (RP-1) or 590 nm (RP-2).

Fluorescent microRNA sensing by CS-ZIF-8-La. The CS-ZIF-8-La nanoparticles (~0.2 mg) were dispersed in 1 mL PBS (10 mM, pH 7.4) containing 40 nM FAM-P2 and 20 nM TAMRA-P2. After 2 h stirring at 25 °C, the nanoparticles (RP-3) were obtained under 11 000 r.p.m. centrifugation and re-dispersed in PBS (1 mL, 10 mM, pH 7.4) for miR-21 sensing. The miR-21 (from 1 to 30 nM) was hybridized with the mixture above for 5 min. Then, the emission spectra were detected under λex = 480 nm (FAM) or 540 nm (TAMRA).

Cell Culture. Hela cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS, all from GIBCO), 5 % penicillin streptomycin glutamine and 5 % sodium pyruvate in a humidified atmosphere containing 5 % CO2 at 37 ºC. Before cell imaging experiment, the cells were incubated in cell culture dishes for 24 h.

Cytotoxicity Assay. The cytotoxicity of CS-ZIF-8-La nanoparticles was tested by MTT assay. Briefly, HeLa cells (1 × 105 cells per well) were seeded and incubated in 96-well plates for 24 h. Then, the original medium was removed, and the cells were incubated with CS-ZIF-8-La nanoparticles (1-80 g·mL-1) for 8 h. After washed with PBS three times, each well was added with MTT solution (0.1 mL, 0.5 mg·mL-1 in PBS) for 4 h incubation. Then, remove the remaining MTT, and add DMSO (150 μL) to dissolve the precipitated formazan violet crystals. Finally, the


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absorbance was determined at 490 nm.

Intracellular miR-21 imaging. Before cell imaging, HeLa cells were incubated for 24 h to obtain a suitable density. Then, the RP-3 (CS-ZIF-8-La nanoparticles hybrid with FAM-P2 and TAMRA-P2) suspension was added to the cell culture wells to get the concentration of ~50 g·mL-1. After incubated for 1-6 h, the DMEM was removed, and the cells were washed three times by PBS (10 mM, pH 7.4) for fluorescent confocal imaging.

RESULTS AND DISCUSSION The ZIF-8 nanoparticles were prepared from methanolic solution of 2-methyl imidazole and ZnAc·2H2O according to the typical procedure.17 Transmission electron microscopy (TEM) and scanning electron microscope (SEM) images show that the monodispersed ZIF-8 nanoparticles are uniform hexagonal nanoparticles with mean sizes of around 300 nm (Figure 2a and Figure S1). Recent reports show that ZIF-8 can bind aptamer-based probes via electrostatic interaction between the phosphate moieties on the aptamer and uncoordinated Zn ions on the surface of ZIF-8.18,19 But ZIF-8 cannot be used as effective quencher for fluorescent “off → on” sensing nucleic acids. The fluorescence quenching capacity of ZIF-8 is resulted from the stacking π-electron interactions of the bridging imidazolate ring and from the zinc ions. Exchanging zinc ions or doping with other transition metal ions, which can effectively suppress fluorescence intensity by static quenching, will be potentially enhance the fluorescence quenching capacity of ZIF-8.20,21 We have found that the trivalent lanthanide ions in NMOFs can be used as quencher for fluorescent DNA ratiometric sensing.16 Therefore, in this study, lanthanide ions (Ln3+) were employed to be doped in ZIF-8 nanoparticles. The Ln3+ doped ZIF-8 (ZIF-8-Lns) nanoparticles prepared following the procedure shown in Figure 2b. The La3+, Ce3+, Nd3+, and Tb3+ were doped into the ZIF-8 respectively. The TEM images (Figure 2c1 and Figure S1) revealed that the as-prepared ZIF-8-Lns retained the size distribution of the starting nanoparticles, but the polyhedral shape was changed or lost. It might because of the ion exchange between the Zn2+ and Ln3+ on the ZIF-8 surface, leading to the changed morphologies. Figure 2c2 shows the high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging as well as the corresponding EDX elemental



mapping of ZIF-8-La, demonstrating that La3+ was not homogeneously distributed throughout the ZIF-8, but mainly on the ZIF-8 surface. The amounts of Zn2+ and Ln3+ in ZIF-8 and ZIF-8-Lns were detected by inductively coupled plasma mass spectrometry (ICP-MS, Figure S2). When the

Ln3+ doped into ZIF-8, 10-13 % of Zn2+ were released due to the ion-exchange.22,23 The X-ray powder diffraction (XRD) patterns in Figure 2d demonstrated the crystallinity of ZIF-8 and ZIF-8-Lns. The crystal pattern of ZIF-8 is identical to the simulated one. While, the ZIF-8-Lns tends to lost their crystallinity. Among the ZIF-8-Lns, ZIF-8-La shows the best diffraction signals, indicating the ZIF lattice is still intact and maintains the crystal structure of ZIF-8. As nanoquenchers, the fluorescence quenching abilities of ZIF-8 and ZIF-8-Lns were evaluated and compared by employing the model dye 2',7'-dichlorofluorescein (DCF). After mixed with ZIF-8 or ZIF-8-Lns, the fluorescence of DCF was quenched with varying degrees. Compared with ZIF-8, ZIF-8-Lns show better fluorescence quenching ability toward DCF. As shown in Figure S3, 83 % of the DCF fluorescence was quenched by ZIF-8; While, there were about 98 % fluorescence quenching by using ZIF-8-Lns. It means the Ln3+ doped in ZIF-8 can enhance the fluorescence quenching ability. All of the ZIF-8-Lns (including ZIF-8-La, ZIF-8-Ce, ZIF-8-Nd and ZIF-8-Tb) show similar quenching intensity toward the fluorescent molecule DCF. Therefore, the ZIF-8-Lns can be potentially used as effective quenchers for DNA or microRNA sensing. The dye-labeled ssDNA (P1, a model chain of tumor suppressor genes) was utilized as model for investigating the fluorescent DNA sensing behavior of ZIF-8-Lns. Briefly, the P1 labeled with fluorophore carboxyfluorescein (FAM) or tetramethylrhodamine (TAMRA) was adsorbed on the surface of ZIF-8-Lns or ZIF-8 via hydrogen bond interactions between the nucleotide bases of P1 and the ZIF nanoparticles. Meanwhile, the emission of FAM or TAMRA was quenched accordingly. After adding the target T1, the dye-labeled P1 hybridized with T1 leading to the formation of double-stranded DNA (dsDNA), which was detached from ZIF nanoparticles. The fluorescence of FAM or TAMRA was simultaneously recovered (Figure 1b and c). Compared with ZIF-8, ZIF-8-Lns exhibited better quenching degree toward the FAM or TAMRA labeled P1. As shown in Figure 2e and Figure S4, ZIF-8 shows no significant affection on the emission intensity of FAM-P1 and TAMRA-P1 after adding T1. But ZIF-8-Lns (including ZIF-8-La, ZIF-8-Ce, ZIF-8-Nd and ZIF-8-Tb) show effective fluorescence quenching ability, and obvious “turn down → turn up” procedure can be observed during the DNA sensing. These results


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are resulted from the intrinsic fluorescence quenching properties of Ln3+. Among the ZIF-8-Lns, ZIF-8-La possesses the best quenching efficiency. It might because the morphology of the ZIF-8-La is better than other ZIF-8-Lns. And, the La3+ ions are mainly distributed on the surface of ZIF-8-La (Figure 2c2), resulting in effective CT happened from the surface adsorbed fluorophores to the Ln ions. At the time of the interaction of FAM-P1 (or TAMRA-P1) with ZIF-8-La, the fluorescence intensity (FI) of FAM, as well as TAMRA, was quenched 88.3 % and 77.5 % respectively. While, on ZIF-8, the FI of FAM and TAMRA was only quenched 29.4 % and 27.9 % respectively. That means the fluorescence quenching ability of ZIF-8-La is around 3 times higher than that of ZIF-8. According to the typical emission of FAM-P1 (Figure 2f and g) and TAMRA-P1 (Figure S5), ZIF-8-La shows obviously “turn down → turn up” procedure for fluorescent T1 sensing compared with ZIF-8 under the same conditions. Furthermore, the concentration ratio of ZIF-8-La and the dye labeled-P1 have an obvious influence on the DNA sensing progress. As shown in Figure S6, when the FAM-P1 and TAMRA-P1 concentrations are below 5 nM containing ~0.2 mg·mL-1 ZIF-8-La in PBS, the emission quenching percentage is over 99 % compared with that of pure FAM-P1 and TAMRA-P1. For aptameric quantitative sensing, the slope of standard curve on ZIF-8-La platform is higher than that on ZIF-8 (Figure S7), indicating higher sensitivity on ZIF-8-La platform. As aptameric sensor, the FAM-P1 or TAMRA-P1 assembled on ZIF-8-La exhibits a linear relationship for the target ranged from 1 to 30 nM (Figure S7 and S8).



Figure 2. Characterizations of ZIF-8 and ZIF-8-Ln, and their fluorescence quenching property toward fluorophore-labeled DNA. (a): TEM image (left) and SEM image (right) of ZIF-8. (b) Scheme of the formation of ZIF-8-Lns. (c) TEM image of ZIF-8-La (c1) and HAADF-STEM elemental mapping of a ZIF-8-La core-shell nanoparticle (c2). (d) XRD patterns of ZIF-8, ZIF-8-Ln and CS-ZIF-8-La. (e) Peak FI of FAM-P1 (20 nM) in the presence or absence of T1 (30 nM) in ZIF-8 or ZIF-8-Lns (~0.2 mg·mL-1, suspended in 10 mM PBS). (f and g) Emission spectra of FAM-P1 (20 nM) mixed with ZIF-8 (f) or ZIF-8-La (g) (~0.2 mg·mL-1) by adding 30 nM T1.

In our previous works, we found the NMOFs can be used as ratiometric sensors for DNA sensing.6,16,24 The surface positive charged property of NMOFs is essential for the emission change toward the positively or negatively charged dye molecules that labeled on DNA. Especially for the negatively charged FAM, the fluorescence of FAM labeled ssDNA can be partly decreased when it incubates with NMOFs, then it will be further decreased after the addition of its target. This phenomenon is attributed to the formed FAM-dsDNA that does not leave away from the NMOFs, but re-interacts with the NMOF surface due to electrostatic


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interaction, resulting in further fluorescence quenching. Therefore, the negatively charged fluorophores (such as FAM) undergo an emission-intensity “turn down → further turn down” procedure. However, the positively charged fluorophores (such as TAMRA) have an emission-intensity “turn down → turn up” procedure. But, in this work, both of FAM and TAMRA fluorophore-labeled DNA experienced the same emission-intensity “turn down → turn up” procedure on ZIF-8 or ZIF-8-La platforms. We suppose there are lack of enough surface metal ions, which are not fully coordinated on the surface of ZIF-8 and ZIF-8-La nanoparticles, resulting in that the FAM-dsDNA cannot be readsorbed on the nanoparticles. The surface charge properties were investigated. It can be seen in Figure 3a, the zeta potentials of ZIF-8 and ZIF-8-La were about 4.75 and 15.38 mV in deionized water respectively. It means the surface positive metals of ZIF-8 and ZIF-8-La are not fully enough for readsorbing the negatively charged fluorophores. For designing ratiometric sensors for DNA or microRNA, more La3+ ions were modified on the surface of ZIF-8 nanoparticles by using a core-shell method. The core-shell nanoparticle (CS-ZIF-8-La) was prepared by modifying a coordination polymer shell, which was prepared by using La3+ and another ligand squaric acid (H2sq), on the surface of the ZIF-8 core. During the preparation of CS-ZIF-8-La, the shell thickness was increased with the increase of the molar ratio of La3+ and H2sq from 0.2 : 1 to 2 : 1 (Figure S9). But when the molar ratio of La3+ and H2sq was 2 : 1, the ZIF-8 core was not stable and collapse. So, we chose La3+ and H2sq with the molar ratio of 1 : 1 for preparing CS-ZIF-8-La. According to the XRD pattern of the CS-ZIF-8-La (Figure 2d), the diffraction peaks still match well with that of the ZIF-8, meaning the structure of ZIF-8 remained in CS-ZIF-8-La. X-ray photoelectron spectroscopy (XPS) was also employed to explore the interactions among 2-methyl imidazole ligand, Zn2+ and La3+ in the prepared CS-ZIF-8-La and ZIF-8-La (Figure 3b and Figure S10). The La3d and N1s peaks in CS-ZIF-8-La were blue shifted by approximately 1.0 eV compared to that in ZIF-8-La, indicating that more La3+ ions were existed in CS-ZIF-8-La. The binding energy of Zn2p in ZIF-8-La was higher than other ZIF nanoparticles, confirming the release of Zn2+ from ZIF-8 precursor during the preparation of ZIF-8-La. While, less Zn2+ ions were released from ZIF-8 core in CS-ZIF-8-La. The morphology of CS-ZIF-8-La was further observed by TEM and HAADF-STEM with corresponding EDX elemental mapping (Figure 3c



and Figure S9). The thickness of the shell of CS-ZIF-8-La was about 20 nm. As shown in Figure 2c2 and Figure 3c2, more La3+ were distributed on the surface of CS-ZIF-8-La compared with ZIF-8-La. The zeta potential of CS-ZIF-8-La was 38.6 mV (Figure 3a), confirming that the La3+ ions on the surface of CS-ZIF-8-La were not fully coordinated. The aptameric sensing behavior of CS-ZIF-8-La was investigated by using P1 labeled with positive or negative charged dyes (such as FAM, Texas red, Cy5 and TAMRA) (Figure S11). Figure 3d exhibits the typical emission spectra of the dyes labeled P1 with the target T1 in different concentrations, by the addition of CS-ZIF-8-La. For Texas red-P1 and TAMRA-P1, the emission intensity “turn down” obviously when the CS-ZIF-8-La were introduced into the aptamer-probe system of 20 nM. After adding the target T1, a strong emission was observed (“turn up”). The “turn up” efficiency corresponds with the T1 concentration. The results are similar as that on earlier reported nanoquenchers (such as graphene oxide, gold nanoparticles and MoS2). And favorable linear correlations can be observed range from 1 to 30 nM (Figure S12). As for FAM-P1 and Cy5-P1, a different emission-quenching behavior was observed on CS-ZIF-8-La (Figure 3d). The emission intensities of FAM-P1 and Cy5-P1 decreased (“turn down”) after mixing with CS-ZIF-8-La respectively. After incubated with T1, the emission of FAM and Cy5 groups was “further turn down”, but the emission of TAMRA-P1 and Texas red-P1 was recovered. By adjusting the adsorbtion amount of FAM-P1 (or Cy5-P1) on CS-ZIF-8-La, linear correlations can also be observed range from 1 to 30 nM (Figure S12).


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Figure 3. Characterizations of CS-ZIF-8-La, and the fluorescence quenching property toward fluorophore-labeled DNA. (a) Zeta potentials of ZIF nanoparticles in water. (b) X-ray photoelectron spectroscopy spectra of ZIF nanoparticles. (c) TEM image of CS-ZIF-8-La (c1) and HAADF-STEM elemental mapping of one CS-ZIF-8-La core-shell nanoparticle (c2). (d) Fluorescence spectra of dye-labeled P1 (40 nM for FAM-P1 and Cy5-P1; 20 nM for TAMRA-P1 and Texas red-P1) mixed with CS-ZIF-8-La (~0.2 mg·mL-1) containing T1 with different concentrations. λex = 480 nm (FAM), 540 nm (TAMRA), 590 nm (Texas red) and 640nm (Cy5) respectively.

Therefore, the CS-ZIF-8-La exhibits completely opposite emission quenching abilities toward negative-dye labeled dsDNA and positive-dye labeled dsDNA. It is because the metal ions (La3+) on the CS-ZIF-8-La surface are only partly coordinated and tend to coordination with the negatively phenolic hydroxyl, charged carboxyl or sulfonic groups on FAM or Cy5, but not tend to interact with TAMRA or Texas red which have positive quaternary ammonium groups (Figure 4a). This coordination interaction effect was characterized by FT-IR employing the FAM-P1 (Figure S13). After FAM-P1 adsorbed on CS-ZIF-8-La, the signals at 1400 and 1139 cm-1 were increased compared with pure CS-ZIF-8-La. The 1400 cm-1 signal was identified as the N-C stretches from the adsorbed nucleotide groups of P1, or from the imidazole ring stretching of CS-ZIF-8-La. And the 1139 cm-1 signal was identified as PO2 stretches from P1, or from the aliphatic N-C stretching of CS-ZIF-8-La.25,26 After hybridization with T1, the signals were further increased, meaning that the formed FAM-dsDNA (FAM-P1 + T1) was not depart from the surface



of CS-ZIF-8-La, but re-adsorbed on CS-ZIF-8-La. The increased signals around 3420 and 3135 cm-1 were from the O-H, N-H and C-H stretches of FAM-dsDNA. While, for TAMRA-P1 + CS-ZIF-8-La, the signals mentioned above (including 1400, 1139, 3420 and 3130 cm-1) were decreased after hybridization with T1. It means that the formed TAMRA-dsDNA (TAMRA-P1 + T1) was left away from the surface of CS-ZIF-8-La. The ratiometric detection of T1 by using CS-ZIF-8-La platform was also monitored by two probes: RP-1 and RP-2 (Figure 4b and c). The RP-1 was prepared by adsorbing FAM-P1 and TAMRA-P1 on CS-ZIF-8-La; and the RP-2 was prepared by adsorbing Cy5-P1 and Texas red-P1 on CS-ZIF-8-La. Both of RP-1 and RP-2 showed obvious ratiometric spectra toward T1. For RP-1, the addition of T1 leaded to the emission of FAM decreased, and the emission of TAMRA increased gradually under the excitation at 480 nm. Similarly, For RP-2, the addition of T1 leaded to the emission of Cy5 decreased, and the emission of Texas red increased under the excitation at 590 nm. Due to the 480 nm and 590 nm are not the maximum excitation wavelengths for TAMRA and Cy5 respectively, there are no dramatical changes of fluorescence intensities of TAMRA and Cy5. The CS-ZIF-8-La platform was also used for aptameric microRNA sensing. The miR-21 is a typical microRNA and has been proved to be an oncogene and antiapoptotic indicator that is upregulated in different kinds of cancer cell lines. The miR-21 aptamers FAM-P2 and TAMRA-P2 were adsorbed on CS-ZIF-8-La as probe (RP-3) for sensing miR-21. Figure 4d and e show obviously ratiometric sensing behavior of the probe toward miR-21. That is, in the presence of miR-21, the fluorescence intensity of FAM decreased, but the fluorescence intensity of TAMRA increased gradually. Good linear correlations between the miR-21 and the maximum emission wavelength of FAM and TAMRA can be observed (Figure 4f and g).


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Figure 4. Ratiometric detection of DNA and microRNA by using CS-ZIF-8-La platform. (a) Illustration of the interaction between the La3+ on the surface of CS-ZIF-8-La and FAM-Labeled dsDNA. (b and c) Fluorescent spectral changes of RP-1 or RP-2 (~0.2 mg·mL-1) containing T1 with different concentrations. λex = 480 nm (RP-1) or 590 nm (RP-2). (d and e) Emission spectra of FAM-P2 (40 nM) (d) or TAMRA-P2 (20 nM) (e) mixed with CS-ZIF-8-La (~0.2 mg·mL-1) containing miR-21 with different concentrations. λex = 480 nm (d) or 540 nm (e). (f and g) Calibration curve for miR-21 detection by assembling 40 nM FAM-P2 (f) or 20 nM TAMRA-P2 (g) with CS-ZIF-8-La (~0.2 mg·mL-1).

The specificity of the nanoprobe RP-3 was investigated by comparing miR-21, miR-141, miR-21-5p, let-7a, ATP, UTP, GTP and CTP at the same concentration. As shown in Figure 5a, the ratio of FTAMRA/FFAM produced by miR-21 was much higher than that induced by other miRNAs or biomolecules. It indicates that the miRNA nanoprobe shows high sequence specificity and demonstrates application prospect in complex biosystems and cellular environment. MTT assays were further employed to investigate the potential cytotoxicity of the CS-ZIF-8-La on living cells. The Hela cells were incubated with 1 to 80 g·mL-1 CS-ZIF-8-La nanoparticles, and the absorbance of MTT at 490 nm was detected to show the activation degree of the cells. Figure 5b shows that treatment with CS-ZIF-8-La nanoparticles induced little reduction in cell viability. Based on the above characterizations, the nanoprobe RP-3 was used for intracellular miR-21 ratiometric probing in Hela cells using confocal microscopy. After the nanoprobe RP-3 was incubated with Hela cells, the intracellular miR-21 interacted with the aptamers FAM-P2 and TAMRA-P2, which have been attached on CS-ZIF-8-La surface. As shown in Figure 5c and d, an obvious emission change can be observed form green (FAM: 500-550 nm) to red (TAMRA: 550-650 nm) under 488 nm excitation wavelength. With the incubation time increasing, the red



color intensities in HeLa cells were responsively increased. The two-color fluorescence images show different amounts and spatial distributions of miR-21 in Hela cells, showing the excellent ability of the nanoprobe RP-3 for imaging intracellular microRNA molecules.

Figure 5. Intracellular miR-21 imaging. (a) The ratio of FTAMRA/FFAM of RP-3 probe toward different biomolecules. (b) Illustration of the miR-21 sensing in cancer cells. (c) Cytotoxicity induced by CS-ZIF-8-La nanoparticles in Hela cells. (d) Confocal images of miR-21 in cancer cells with different incubation time (1-6 h). Scale bars: 50 μm.

CONCLUSION In summary, La3+ doped in ZIF-8 can enhance the fluorescence quenching ability of ZIF-8, affording an ideal fluorescent sensing platform for DNA detection. The La3+ in ZIF-8-La can reduce the background fluorescence, resulting in increased signal-to-background ratio around 3 times higher than pure ZIF-8. The signal-to-background ratios shows an ideal linear relationship with the concentration of analyte (DNA or microRNA) ranged from 1 to 30 nM. Furthermore, the core-shell CS-ZIF-8-La, simply prepared at room temperature, exhibited the same fluorescence sensing behavior toward positive-dye labeled DNA (TAMRA-P1 and Texas red-P1), but showed completely contrary fluorescence quenching behaviors toward negative-dye labeled DNA (FAM-P1 and Cy5-P1). By using CS-ZIF-8-La as quenching platform, ratiometric sensors were designed for fluorescent detecting DNA and microRNA. This work will more widely expand the application of ZIF-8 in the field of biomolecule sensing.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental details and data; Figures S1−S13.

AUTHOR INFORMATION Corresponding Author * [email protected]; [email protected]

Author contributions ⊥

The first two authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21705165), and "Double First-Class" university project (No. CPU2018GF07).

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Regulating Fluorescent Aptamer-Sensing Behavior of Zeolitic

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