Enhancing the Stability of Cu‐BTC Metal‐Organic Framework via the Formation of Cu‐BTC@Cu3(PO4)2 MOF Core‐Shell Nanoflower Hierarchical Hybrid Composites

Hybrid organic‐inorganic nanoflowers (NFs) have recently emerged as a critical tool in enhancing the stability and activity of biomolecules due to their expansive surface area and porosity. The delicate petal‐like features of NFs offer innumerable sites for biomolecule adsorption, including but not limited to proteins, amino acids, and enzymes. Cu‐BTC, a copper‐based Metal‐Organic Framework (MOF) has been hindered in its potential for diverse applications by its instability in humid and aqueous conditions. To overcome this limitation, this study explores the stabilization of Cu‐BTC via the mineralization of its surface with the formation of copper phosphate nanoflowers (NFs). To initiate the mineralization process and provide a template for the growth of the NFs, a physiologically rich amino acid medium is employed. The inclusion of amino acids in the RPMI medium played a crucial role in the preservation of the Cu‐BTC hierarchical structure by facilitating the self‐assembly of copper phosphate nanoflowers on its surface, thereby producing a Cu‐BTC@Cu3(PO4)2 core‐shell structure. The innovative mechanism behind the formation of copper phosphate nanoflowers in this study and its consequential stabilization of the Cu‐BTC MOF structure underscore its novel nature.

soy protein, [33] biosurfactants, [34] glucose oxidase, and catalase, [35] silk-fibroin protein [36] and amino acids [37,38] were used to facilitate the growth of the NFs. NFs were also synthesized using amino acids as the organic component and metal phosphates or metal ions as the inorganic material. [37,38] Wu et al. reported that CP NFs formation and growth were more selective to positively charged amino acids than neutral and negatively charged ones. [39] The CP NFs grown onto Lysine (Lys) exhibited a more compact structure than the NFs grown on Asparagine (Asn). [39] Further, the CP NFs-Lys hybrid structure revealed a higher peroxidase-like activity than the CP NFs-Asn counterparts. [39] Lee et al. revealed the three-step process of NFs formation: 1) the formation of primary crystals of metal phosphate from the coordination of metal ions with proteins through amide groups, 2) nucleation of metal-protein crystals into larger molecules with petals starting to form, and 3) protein-mediated crystal growth to form the complete structure while keeping the scaffold intact. [16] The aim of this study is to assess the structural and morphological integrity of Cu-BTC nanostructures under two different media conditions, PBS (amino acid-free) and RPMI (amino acid-rich), while investigating the potential transformation of Cu-BTC into NFs. The released Cu 2+ ions from the Cu-BTC MOF will form complexes with phosphate ions present in the physiological media, thereby promoting the growth of nanoflowers onto the surface of the MOF. This strategy aims to prevent the disintegration of the structure and maintain its overall stability.

Results and Discussion
The PXRD pattern for Cu-BTC (see Figure 1a) is consistent with previously published work. [25,26,37] The material exhibited high crystallinity with intensive peaks appearing in the 2θ range of 5° to 15°, where the most intense peaks were observed at 2θ values of 5.97, 6.88, 9.67, 11.80, and 13.58°. The presence of high intensity XRD peaks within this range is characteristic of microporous MOF materials due to the small pores and cavities within their hierarchical structure. All peaks were indexed to their standard hkl values as compared with the previously published work. [25,26,37] The inset of Figure 1a shows the paddlewheel characteristic building unit of Cu-BTC MOF where two Cu 2+ ions are bridged through four carboxylate anions. [40] The Cu-BTC FT-IR spectrum shown in Figure S1 exhibits vibrational bands at 1639, 1445, and 1378 cm −1 representing the vibrations of the COOH groups from the BTC linker. The low-intensity, broadband at 3500 cm −1 indicates the complete deprotonation of the BTC carboxylic acid groups and attributed to the physically adsorbed water molecules. The linkage between Cu 2+ ions and the BTC carboxylate anions were shown as bands representing the Cu-O group at 752.2 and 515.5 cm −1 . The intense bands in the FT-IR spectrum of the as-prepared Cu-BTC further confirm its high crystallinity.
Cu-BTC exhibits a defect-free octahedral crystal structure with sharply identified edges and an overall unified morphology and size distribution, as shown in the SEM micrograph of the as-prepared Cu-BTC in Figure 1b. The elemental analysis of the Cu-BTC powder was further examined for its elemental composition, as shown in the EDX pattern in Figure S2, Supporting Information. The presence of C, O, and Cu with weight percentages of 16.23, 19.33, and 35.86, respectively, indicates the compositional purity of the prepared Cu-BTC. The Langmuir surface area and the Brunauer-Emmett-Teller (BET) surface areas were 1507 m 2 g −1 and 1268.9 m 2 g −1 , respectively. These findings are in accordance with the literature and confirm the high porosity of the MOF structure.
To investigate the structural and morphological stability of Cu-BTC, PBS and RPMI media were used to measure the release of Cu 2+ ions from the structure at different time points (see Figure 2). A burst release of Cu 2+ ions was observed in both media; however, the rate of increase of Cu 2+ ions in PBS solution was six times higher than that in RPMI media within the first 3 h. The initial leaching out of Cu 2+ ions indicates the breakdown of the hierarchical Cu-BTC structure, owing to its role as a drug delivery vehicle. [41] After the first 3 h, the inhibited leaching of Cu 2+ ions in the RPMI media was evident. This inhibition could be due to the presence and adsorption of the amino acids onto the surfaces of the Cu-BTC particulates. The highest observed concentration of Cu 2+ ions released in the RPMI media was 18 ppm compared to 145 ppm in PBS. Throughout the entirety of the  Figure 1a is the paddlewheel crystal structure of Cu-BTC MOF. The high crystallinity and distinct morphology of the Cu-BTC confirms its phase purity.

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3-days experiment, the degree of Cu-BTC dissolution and the resultant liberation of Cu 2+ ions remained consistent across both media.
An investigation of the structural and morphological integrity of the Cu-BTC particulates after 24, 48, and 72 h of exposure to both types of media was carried out. Figure 3 reveals the XRD patterns of the dried Cu-BTC particulates upon treatment with PBS and RPMI at different time periods. The burst release of Cu 2+ ions from the Cu-BTC particulates in PBS media was reflected in a dramatic change in the phase composition and crystallinity of the Cu-BTC structure, as shown in Figure 3a. The most intense peaks that characterize the structure of Cu-BTC as a MOF nanostructure at 2θ values 5-15° were diminished, leaving more intense peaks at 2θ values of 30.2, 31.2, 35.2°, and 41.2°. These results indicate the destruction of the Cu-BTC structure and its conversion to mixed Cu oxide (Cu 2 O and CuO) phases as well as Cu-coordinated BTC units. [42] Alternatively, the XRD patterns of the Cu-BTC particles treated with RPMI (see Figure 3) revealed a preserved crystallinity and phase composition of the structure for all time points. All Cu-BTC characteristic XRD peaks were observed in the XRD patterns after 24, 48, and 72 h. However, new peaks were observed after a 48 h incubation period and further increased in intensity after 72 h of exposure, as shown in Figure 3b. These peaks appeared at 2θ values of 9.3, 9.9, 10.9, 15.1, 17.1, 18.6, 21.9, 25.3, and 28.5° and are attributed to the presence of the Cu 3 (PO 4 ) 2 crystalline phase, according to the JCPDS card number 80-0992. [43] The effect of treatment of Cu-BTC particulates in PBS and RPMI media for 72 h on the composition of the treated Cu-BTC was further examined using FTIR, Raman spectroscopy, and TGA-DTG analysis. The FTIR spectra in Figure 4a compare the 72 h treated samples with the as-synthesized Cu-BTC. Comparing to Cu-BTC FTIR results, the FTIR spectrum of the RPMItreated samples exhibited the same bands with lower intensity and more broadness. Three bands were observed at 1050.9, 560, and 630 cm −1 , which are attributed to the stretching absorption of the P-O bond of a phosphate group. Further, two bands were observed at 990, and 1050 cm −1 , which are attributed to the absorption of the Cu-OH bond. A high intensity and broadband was also observed at 1577 cm −1 , which is related to the presence of an amide group. Moreover, a group of high-intensity broad bands were observed at 3550, 3439, and 3217 cm −1 , attributing to the O-H stretching absorption of physically adsorbed water, at 3550 and 3439 cm −1 , and water of hydration, at 3217 cm −1 . The existence of water hydration may be linked to the emergence of Cu 3 (PO 4 ) 2 , a freshly produced compound that typically manifests as Cu 3 (PO 4 ) 2 .3H 2 O due to its inherent hydration properties. Further, the presence of a strong band at 1620 cm −1 confirms the presence of water crystallization. The PBS-treated Cu-BTC exhibited a disappearance of the BTC characteristic bands at 1639, 1445, and 1378 cm −1 and those representing Cu-O bonds. Also, a high-intensity broadband was observed at 3400 cm −1 , which could be attributed to the presence of physically adsorbed water onto the newly formed Cu oxides. These results suggest that the formation of Cu-coordinated BTC units is marginally possible as a result of the treatment of the Cu-BTC  The Raman spectrum of the pristine Cu-BTC MOF shown in Figure 4b reveals the presence of high-intensity sharp peaks at 1007.9 and 1616.6 cm −1 , which are attributed to the aromatic CC group of the BTC linker. [44] In addition, doublet peaks at Raman shifts of 748.2, 829 cm −1 , and at 1463.3, 1544.4 cm −1 are attributed to the CH and CO functional groups, respectively. [44] A low-medium intensity peak was observed at 501.3 cm −1 , relating to the CuO linkage. [44] The RPMI-treated Raman results indicate the presence of the same peaks, confirming the intact structure of Cu-BTC. In addition, new peaks were observed at 440.7 cm −1 (PO bond), 1435.3 cm −1 (CH 2 group), and 1578.3 and 1587.5 cm −1 (amide groups). These new peaks confirm the presence of phosphate and amide groups onto the structure of the RPMItreated Cu-BTC absent in the Raman spectrum of the PBStreated Cu-BTC. The PBS-treated results show an increase in intensity and sharpness of a peak at 590 cm −1 confirming the high possibility of the presence of Cu oxides in the produced solids.
The TGA and DTG thermograms of the as-prepared Cu-BTC and the PBS-and RPMI-treated Cu-BTC for 72 h can be seen in Figure 5. Pure Cu-BTC displayed two main thermal events at 64.6 and 335.5 °C, attributing to the evaporation of physically attached water molecules and the thermal degradation of the BTC linker and the consequent evaporation of the organic degradation products. [45] The extent of water vapor removal accounts for a weight loss of 13.2%, while the thermal breakdown of the BTC linker accounts for a weight loss of 33.7%. Lower extent weight loss events were also observed throughout the diagram, which can be attributed to the removal of volatile organic residues. The overall weight loss of the Cu-BTC was estimated to be 66.8%. Inorganic CuO residue was formed in the process and is evident by the continuing plateau after 500 °C. [45] RPMI-treated Cu-BTC exhibited more thermal events indicative of the removal of physically and chemically attached water with weight losses of 15.3 and 9.8% and 92.2 and 167.8 °C, respectively. These events were followed by more extensive weight loss events at 307.9 and 419.7 °C, which are attributed to the formation of the dehydrated Cu 3 (PO 4 ) 2 phase and the degradation of the BTC
The delay in the thermal event of the BTC unit suggests the newly precipitated Cu 3 (PO 4 ) 2 phase on the structure in contrast the TGA and DTG thermograms of the PBS-treated Cu-BTC shows an overall weight loss of 13.3% which combines three weak thermal events at 69.7, 210.2, and 391.6 °C. These events represent the removal of the physically and chemically adsorbed water molecules as well as the breakdown of the remaining BTC units. The plateau after 400 °C corresponds to the formation of the CuO residue in the sample.
The variation in the morphology of the Cu-BTC crystallites as a result of treating them with PBS and RPMI for up to 72 h can be seen in Figure 6. The octahedral morphology of the pristine Cu-BTC was clearly destructed in PBS media (Figure 6 a-c). An agglomeration of the irregularly shaped Cu carboxylate and Cu oxide remains is revealed with no signs of Cu-BTC octahedron crystallites. However, the micrographs shown in Figure 6 d-f indicate the presence of highly porous nanotextured flowers with nano features onto the surfaces of the MOF crystallites, producing a hierarchical Cu-BTC MOF@nanoflower core-shell composite structure. The extent of formation of the nano-textured flowers is increased with increasing treatment time from 24 to 72 h. A closer look at the morphology of an individual nanoflower grown onto an individual Cu-BTC crystallite, when treated with RPMI media, is shown in Figure 7. The newly formed nanotextured flowers are highly porous and spherical in shape, consisting of thin leaflets (approximately 100 nm in thickness), that have been epitaxially grown onto the Cu-BTC crystallites, as demonstrated in Figure 7a. Figure 7b Figure S3, Supporting Information, illustrates EDX mapping of Cu-BTC compared to the leaflets of a nanoflower. Cu and O can be seen in both the MOF and microflower structures with P covering most of the microflower and only slightly present on the Cu-BTC crystallite. Moreover, the presence of N was also observed in the entire morphology of the nanotextured flowers, as shown in Figure S3b. These results confirm the elemental analysis of the nanotextured flowers and indicate the adsorption of amino acids from the surrounding RPMI media onto the surfaces of the Cu-BTC crystallites.
A proposed mechanism for the formation of the copper phosphate nanotextured flowers onto the surfaces of the Cu-BTC crystallites is shown in Figure 8. The leaching out of Cu 2+ ions results in the partial destruction of the Cu-BTC 3D structure and the presence of surface micro and nano cracks onto the surfaces of the Cu-BTC crystallites. In the presence of RPMI, the ions react with the PO 4 3− ions in media precipitating and forming primary crystals of copper phosphate.
Copper phosphate seeds epitaxially grow onto the surface of the Cu-BTC crystallites where nucleation happens from the amino acids in media (see Figure S4a, Supporting Information). Precipitation and subsequent self-assembly of the leaflets are facilitated by the amino acids mainly through the coordination of the amide groups and the metal ion. RPMI media contains 20 amino acids with various concentrations (see Table 1). Previously, Wu et al. determined that positively charged amino acids highly impacted the nucleation and growth process of the nanotextured flowers. [39] Therefore, the presence of L-Cystine and L-Arginine in the RPMI medium are considered to play a crucial role in the growth of copper phosphate nanotextured www.advmatinterfaces.de flowers. Further, the initial dissolution of the Cu 2+ ions from the MOF structure is concurrent with the adsorption of the amino acids on the surface. This results in a roughness of the surfaces of the crystallites (see Figure 7a and Figure S4b, Supporting Information).
The high porosity and interconnectivity of the leaflets are evident in Figure S4b, Supporting Information. The mechanism shown in Figure 8 indicates the self-assembly of the nanotextured flowers resulting in complete coverage of the Cu-BTC crystallites and the formation of a core-shell structure. The stabilized Cu-BTC crystallites offer a durable platform for various applications, including the creation of sensors. In this context, the stabilized Cu-BTC, which features multifaceted nanoflowers possessing a combined greater surface area than pristine Cu-BTC, has the potential to function as an independent sensor or as a component in compos-ites that incorporate other nanomaterials suitable for these applications. [47,48]

Conclusion
In this study, we reported the development of a hybrid organicinorganic NFs onto a copper-based MOF generating a hierarchical core-shell composite, thus enhancing the stability and structural integrity of Cu-BTC. Importantly, this innovative and novel approach provides an effective means of stabilizing Cu-BTC in aqueous media, while offering a highly porous hierarchical structure for a diverse range of applications, including but not limited to enzyme immobilization, drug delivery, and sensing. The nanotextured flower-like crystals of copper phosphate exhibit a regulated growth on the surfaces of Cu-BTC,

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which is attributed to the positively charged amino acids present in RPMI media. Conversely, in amino acid-free media, Cu-BTC undergoes complete structural breakdown due to the breakdown of Cu-BTC linkages. The uncontrolled and rapid release of Cu 2+ ions from the MOF structure was effectively inhibited, thereby sustaining the release for up to 72 h. The formation of Cu 3 (PO 4 ) 2 on the MOF material has the potential to enhance intrinsic peroxidase activity, increase enzymatic activity, and enable sustained release of copper ions from the MOF structure. Moreover, the intricate and highly porous composite structure holds promise for sensing applications that rely on materials with a high surface area. Cu-BTC has garnered attention for its remarkable anti-cancer, anti-bacterial, and sensing properties. Our forthcoming research endeavors aim to assess the performance of the hierarchical Cu-BTC@Cu 3 (PO 4 ) 2 core-shell nanoflower composite in these applications, with the goal of determining its superiority in comparison to the free Cu-BTC MOF.

Experimental Section
Materials: Copper (II) nitrate hemipentahydrate, 1,3,5-benzenetricarboxylic acid, phosphate buffer solution, and RPMI media were purchased from Sigma-Aldrich. N,N-dimethylformamide, dichloromethane, and ethanol were purchased from Fisher. Ultrapure water was obtained from a Millipore pure water system. All chemicals were used without further purification.

Synthesis and Characterization of Cu-BTC MOF:
The preparation of Cu-BTC MOF followed the procedure outlined by Rowsell and Yaghi. [49] Typically, 1.0 g (4.76 mmol) of BTC and 1.72 g (8.62 mmol) of the Cu 2+ salt were each dissolved in 24 mL of solvent consisting of equal parts DMF, ethanol, and deionized water. Subsequently, the Cu 2+ solution was added to the linker solution and stirred for 15 min. The entire mixture was added to a 150 mL sealed tube and placed in an oven at 85 °C for 24 h. After decanting the hot mother liquor and rinsing with DMF, the product was immersed in DCM and filtered, washed, and replaced with fresh DCM daily for 3 days. The washed Cu-BTC MOF crystallites were dried under vacuum at 140 °C for 24 h to remove the remaining solvents and activate the powder.
Characterization of Cu-BTC MOF: The phase purity and crystallinity of the Cu-BTC MOF prepared in this study were evaluated using powder XRD. PXRD data was recorded on a Shimadzu-6100 powder XRD diffractometer with Cu-K α radiation, λ = 1.542 Å. Diffraction data were collected in the 2θ angle range of 20-70 degrees. The morphology and elemental analysis of the Cu-BTC MOF sample were obtained using a ThermoFisher Scientific -Quatro S. BET and Langmuir surface areas of the Cu-BTC MOF samples were determined from N 2 adsorption isotherms at 77 K on a Quantachrome Autosorb-1 volumetric gas sorption instrument. The Cu-BTC MOF material was first degassed at 140 °C for 3 h to completely eradicate all solvents and moisture from the pores. Fourier transform infrared (FTIR) spectra (4000-400 cm −1 ) was obtained from KBr pellets using a Bruker Vector 22 instrument.
Evaluation of Cu-BTC in PBS and RPMI Media: 100 mg of the Cu-BTC powder sample was added to 10 mL PBS or RPMI media in a polyethylene sample tubes. All sealed tubes were placed in a shakerincubator at 110 rpm at 37 °C for up to 72 h. The supernatant was collected after 1, 3, 6, 9, 12, 24, 36, and 48 h, filtered through a 0.22 µm syringe filter and analyzed using an inductively coupled plasma-atomic Table 1. Composition of RPMI -types of amino acids with highest concentrations. An emphasis is given to positively charged amino acids that have been proven to provide templating surfaces for the nucleation and growth of copper phosphate nuclei and nanoflowers, respectively. ]. The experimental results were graphed as a function of time, and the rate of Cu 2+ ion release from Cu-BTC powder following immersion in PBS and RPMI media was determined as a measure of Cu-BTC degradation in these aqueous environments. Solid samples were subjected to drying and characterized using XRD, FT-IR, and Raman spectroscopy over time. TGA was employed to determine the thermal history and composition of Cu-BTC treated in PBS and RPMI for 72 h in comparison to the pristine Cu-BTC. The morphologies of Cu-BTC powders exposed to PBS and RPMI for 24, 48, and 72 h were assessed using the SEM-EDX technique, with both EDX spectra and mapping obtained for Cu-BTC after 3 days of immersion in these media.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.