Gallic acid nanoflower immobilized membrane with peroxidase-like activity for m-cresol detection

We report fabrication of new generation nanoflowers (NFs) using gallic acid (GA) and copper (II) ions (Cu2+) acted as an organic and inorganic component, respectively with effective peroxidase mimic activities in solution and on filter membrane. Unlike the typical protein NFs synthesis mechanism, gallic acid NFs (GA-NFs) was formed via coordination reaction between carboxyl groups of GA and Cu2+. The different morphologies of the GA-NFs were acquired based upon whether the carboxyl groups in gallic acid are active or not. The peroxidase mimic activity of the GA-NFs relied on the Fenton reaction in the presence of hydrogen peroxide (H2O2) was tested towards m-cresol as a function of concentration of the GA-NFs, m-cresol, H2O2 and reaction time. Under the optimized conditions, the oxidative coupling of m-cresol with 4-aminoantipyrine (4-AAP) was catalyzed by the GA-NFs dispersed in solution and adsorbed on filter paper to form an antipyrine dye and it was visually and spectrophotometrically recorded. The m-cresol with range of 0.05–0.5 mM was detected in 10 min and 15 min by using the GA-NFs in solution and on filter paper, respectively. We demonstrated that the NFs can be produced from non-protein molecules and GA-NFs can be used as a promising nanocatalyst for a variety of applications.


Results
Synthesis of GA-NFs. The synthesis of organic-inorganic nanoflowers (NFs) relies on coordination reaction between amide groups in the protein backbone and Cu 2+ in Cu 3 (PO 4 ) 2 primary crystals. Although, nonprotein molecules including amino acids (known as the building blocks of proteins), catecholamines or some model plants extracts acted as organic components of the NFs, almost all recent studies have taken the coordination between the amide group and Cu 2+ as a key step for formation of the NFs. It seems that the selection of amide group containing molecules have become a mandatory in NFs synthesis, which can be considered as a major disadvantage of the NFs formation.
Herein, we present, for the first time, an inspirational work with fabrication of gallic acid incorporated nanoflower (GA-NFs) by exploiting coordination reaction occurred between carboxyl group of GA and Cu 2+ . The typical NFs synthesis procedure was applied that free GA was added into 10 mM PBS solution containing Cu 2+ , and then the resulting mixture was left for incubation without disturbing. The carboxyl group of GA reacted with Cu 2+ in Cu 3 (PO 4 ) 2 primary crystals to initiate the formation of GA-Cu 3 (PO 4 ) 2 complexes as seeds. Interestingly, GA-Cu 3 (PO 4 ) 2 complexes were grown as large petals and these GA-incorporated petals bound to each other by acting as a glue, as protein incorporated ones acted in discovery of NFs before. Then, complete flower-shaped structure called "nanoflower (NF)" is occurred with saturation of anisotropic growth. The formation mechanism of GA-NFs was demonstrated in Scheme 1 step by step.
As an interesting and worthy approach, the roles of activation of carboxyl group on the morphologies of GA-NFs were systematically examined. The 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in standard protein labeling chemistry or called "EDC/NHS chemistry" was utilized to activate the carboxyl group of free GA. Then, typical NF synthesis procedure was followed to show how activated carboxyl group influence morphology of GA-NFs.
Structure of the GA-NFs with peroxidase mimic activities in solution and on filter membrane were characterized and interpreted. In our system, the GA-NFs were dispersed in solution and physically adsorbed on filter membrane for detection of m-cresol known as important hazardous compound, by UV-Vis spectrophotometer and naked eye (Fig. 1A). The oxidative coupling reaction between m-cresol and 4-AAP catalyzed by GA-NFs in the presence of H 2 O 2 was shown in Fig. 1B. Characterization of GA-NFs. The structure of GA-NFs was characterized via several methods. The morphologies (shape, size and surface property) of GA-NFs and carboxyl group activated GA-NFs (cGA-NFs) were monitored by scanning electron microscopy (SEM). The SEM images in Fig. 2A, B show that GA-NFs are spherical with ~ 4 µm size. It seems that the GA-incorporated large petals have plate like shapes, and they have vertically inserted each other to form the GA-NFs. The small spheres (shown in black square) on surface of magnified GA-NFs image in Fig. 2B can be indication of newly occurred GA-Cu 3 (PO 4 ) 2 nanocrystals. We hypothesize that formation of the GA-NFs may be kinetically slow and under continuous formation process. Figure 2C shows that when reaction time was prolonged, the small spheres grew and wrapped surface of the GA-NFs as belts (shown in black rectangular). With the worthy approach, we activated carboxyl group of GA to facilitate coordination reaction between GA and Cu 2+ . And then, the ~ 9 µm sized, uniform, and mono-dispersed cGA-NFs with highly porous structure were produced as shown in Fig. 2D. The high-magnification image of the cGA-NFs was also presented in inset of Fig. 2D.
As a further structural analysis, Energy Dispersive X-ray Analysis (EDX) was used to show the presence of Cu metal in the Cu 3 (PO 4 ) 2 scaffold (Fig. 3A). As the formation mechanism of the NF have been well documented, metal ions, especially Cu 2+ acts as an indispensable cornerstone component for building of the NF. The bending and stretching in the cGA-NFs were evaluated with Fourier-transform infrared spectroscopy (FTIR) as shown in Fig. 3B. For analysis of free GA (blue line), the stretching of O-H groups at 3268 cm -1 , strong absorption of COOH (carboxylic acids) at 1606 cm -1 and C=O stretching at 1467 cm -1 are attributed to characteristic peaks of free GA. The characteristic peaks of 1029 cm -1 and 555 cm -1 refer to PO 4 3− vibrations of Cu 3 (PO 4 ) 2 as given with red line 15 . While the PO 4 3− vibrations in cGA-NFs are assigned to 1038 cm -1 and 558 cm -1 , the stretching of O-H group with different mode, moderate absorption of COOH and C=O stretching are observed at 3419 cm -1 , 1623 cm -1 , 1469 cm -1 , respectively. The consistency in the FTIR spectra is an indication of cGA incorporated NFs. Detection of m-cresol. The m-cresol and its s isomers o-cresol and p-cresol can be enzymatically oxidized but with different efficiencies owing to favorable position of ring substituents in their structures 21,22 . We chose m-cresol for catalytic reaction due to high substrate specificity at room temperature (RT: 20 °C). After the synthesis of cGA-NFs, we demonstrated carboxyl group activated cGA-NFs exhibited much enhanced peroxidase mimic activity compared to GA-NFs formed of GA molecule containing non-activate carboxyl group. The reasons for morphology dependent activity can be attributed to highly porous and compact structures of cGA-NFs. As shown in Figure S1A, the cGA-NFs display higher peroxidase like activity to catalyze the reaction between 4-AAP and m-cresol in the presence of H 2 O 2 than GA-NFs. Then, we only used cGA-NFs in all peroxidase mimic activity experiments. The cGA-NFs acted as a Fenton reagent in the presence of H 2 O 2 and then exhibited peroxidase mimic activity through Fenton reaction. The potential mechanism for Fenton reaction is that Cu 2+ ions in the cGA-NFs react with H 2 O 2 to produce Cu 1+ . Followingly, interaction between Cu 1+ and H 2 O 2 resulted in highly reactive hydroxyl radical (·OH), which catalyzes oxidative coupling reaction between m-cresol and 4-AAP to form an antipyrine dye as a colored compound. The mechanism for the potential Fenton-like reaction is given in Eq. (1).
The peroxidase mimic activities of the cGA-NFs were evaluated in solution and on filter membrane against m-cresol under the various experimental parameters. In Fig. 4, the peroxidase mimic activity of the cGA-NFs dispersed in solution was studied. A standard activity protocol was followed; oxidative coupling between m-cresol and 4-AAP was catalyzed by the cGA-NFs with intrinsic peroxidase-mimic activity. The cGA-NFs (0.5 mg/mL) was added into solutions containing 4 mM 4-AAP, 1 mM H 2 O 2 and m-cresol with a series of concentrations (0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM) and each solution was incubated for 10 min, then its activity was tested in each solution as a function of m-cresol concentrations (Fig. 4A). It reveals that absorption values and color intensity (the direction of the arrow in the photo is from high concentration to low in Fig. 4A) of the product solutions were increased with increase in m-cresol concentrations. The same activity protocol in  www.nature.com/scientificreports/ incubated for 10 min, 30 min, 60 min and 90 min. The absorbance of each solution was measured by UV-Vis. Figure 4B shows that although intense color change and high absorbance values after catalytic reactions was obtained in long incubation times (90 min and 60 min), in 10 min or even in 5 min (data now shown) the remarkable color change and absorption increase were observed (the direction of the arrow in the photo is from high incubation time to short in Fig. 4B). The effect of presence of H 2 O 2 was shown in Supplementary Information Figure S2. It reveals that the efficiency of oxidative coupling reaction between 4-AAP and m-cresol was enhanced with H 2 O 2 . To demonstrate the activity and stability of the cGA-NFs, the reaction solution containing 0.5 mg/mL cGA-NFs, 0.4 mM m-cresol, 4-AAP and 1 mM H 2 O 2 , was applied successive catalytic use (Fig. 4C). We clearly showed that the cGA-NFs lost 60% its initial catalytic activity over the six cycles, which may exhibit high catalytic performance and stability. SEM images of the GA-NFs were recorded before reaction (left one in Fig. 4D) and after the six cycles (right one in Fig. 4D). It is clear that the morphology of the GA-NFs was slightly distorted after six cycles in use compared to intact cGA-NFs image.
To use the cGA-NFs as an attractive nanobiocatalyst, we non-covalently deposited the cGA-NFs on the surface of commercial filter membrane, then investigate how it exhibit peroxidase mimic activity as function the cGA-NFs, m-cresol and H 2 O 2 concentration and reaction time. As a first parameter (Fig. 5A), a series concentration of the cGA-NFs was absorbed on filter membranes, then reaction solution (4 mM 4-AAP, 1 mM H 2 O 2 and 0.4 mM m-cresol) was injected each membrane to observe its activity in 15 min. It is noticed that using the filter membrane high amount of the cGA-NFs exhibited much efficient catalytic activity, which is quite consistent with absorption value and color intensity of the product solution (the direction of the arrow in the photo is from low amount cGA-NFs to high on filter membrane in Fig. 5A). We realized that 2 mg/mL cGA-NFs adsorbed filter membrane can be ideal nanobiocatalyst for further reaction owing to effective peroxidase mimic activity. As aforementioned above, presence of H 2 O 2 and its concentration are vitally important for rapid and efficient catalytic activity as shown in Fig. 5B. We fixed the concentration of the cGA-NFs on filter membrane, m-cresol and 4-AAP to be 2 mg/mL, 0.4 mM and 4 mM, respectively, then concentrations of H 2 O 2 were varied. Although  Figure 5C shows that while even lowest concentration of m-cresol (0.05 mM) was oxidized by the filter membrane and spectrophotometrically and visually detected but in 60 min, however, almost the same catalytic performance was obtained when using 0.5 mM and 0.4 mM m-cresol. The recycling of the filter membrane was tested over the six catalytic cycles, the filter membrane maintained almost 60% of its first cycle activity even after six cycles (Fig. 5D). We hypothesize that favorable conformation of the cGA-NFs can be slightly changed after third cycle was, then gradual reduction in catalytic activities were observed after third cycle. And, the distance between petals or layers of cGA-NFs can decrease and they may stick each other, then catalytic activity can be adversely influenced after third cycle wash. We claim that the cGA-NFs adsorbed on filter membrane possessed much durability compared to the cGA-NFs dispersed in solution, as how the enzyme NFs show enhanced stability compared to free enzymes. We also monitored how successive catalytic reactions influence the morphologies of the cGA-NF on filter membrane with SEM images. The SEM image of the filter membrane were obtained after first and six cycles as seen on top of blue column (image after first cycle) and orange column (image after six cycles), we analyzed that no remarkable distortion on both SEM images. The potential reasons for reduction in catalytic activity of the filter membrane after repeated use can be i)adsorption of excess m-cresol on surface of the cGA-NFs may increase mass-transfer limitations and ii) excess m-cresol may attack available or accessible Cu 2+ ions and form m-cresol-Cu 2+ complexes, both of which may obstruct catalytic activity performance of the NFs. it is worthy to point out that although the morphology of the NFs is not significantly altered or impaired, thecatalytic activity decreases due to the reasons mentioned above. www.nature.com/scientificreports/

Discussion
In summary, we have developed cGA-NFs as new generation NFs and investigate their peroxidase mimic activities through the Fenton reaction when dispersed in solution and adsorbed on filter membrane towards m-cresol. In synthesis procedure, carboxyl group of GA molecules reacted with Cu 2+ for formation of cGA-Cu 3 (PO 4 ) 2 primary nanocrystals, then cGA-NFs was kinetically formed. We also activated carboxyl group of GA, then we showed how it influences morphology and peroxidase mimic activity of the cGA-NFs. The peroxidase-mimic activities of the cGA-NFs in solution and on filter membrane was optimized under various experimental parameters. The both synthesis of the cGA-NFs and the preparation of cGA-NFs deposited filter membrane opened up new avenue in designing novel biocatalytic system. The uniform, mono-dispersed and porous cGA-NFs with intrinsic peroxidase-mimic activity can be promising alternative to enzyme-incorporated NFs and find widespread use in various scientific and technical fields.    16 .Gallic acid was used as an organic part and Cu 2+ ion acted as an inorganic part for the synthesis of GA-NF. Briefly, 0.02 mg/mL gallic acid was dissolved in distilled water. An aqueous solution of CuSO 4 (120 mM, 660 µL) and gallic acid was added to 100 mL phosphate buffer saline (PBS) solution (10 mM, pH 7,4). The mixture was stirred vigorously for 5 min to increase interaction between Cu 2+ and gallic acid. After the incubation at 25 °C for 3 days without disturbing, the precipitates formed at the bottom of the solution were collected through centrifugation (5000 rpm, 10 min) and washed with pure water several times. The obtained product was dried at 50 °C. For the synthesis of cGA-NFs, 0.02 mg/mL was dissolved in PBS solution (10 mM, pH 7.4). The solution was mixed with EDC (10 mM) and NHS (12 mM), following by stirring overnight at room temperature. Then an aqueous solution of CuSO 4 (120 mM, 660 µL) was added to mixture and incubated at 25 °C for 3 days. The obtained products bottom of the solution were collected through centrifugation (5000 rpm, 10 min) and washed with pure water several times. The cGA-NFs was dried at 50 °C.

Detection of m-cresol in solution.
Different concentration of m-cresol (0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM and 0.5 mM) was added to PBS solution (0,1 M pH 7,4) containing cGA-NFs (1 mg/mL), H 2 O 2 and 4-AAP (4 mM). These solutions were incubated at room temperature for 15 min, followed by centrifugation at 8000 rpm for 5 min to remove cGA-NFs. The absorbance of resulting solutions was measured by UV-Vis spectrophotometer.
Detection of m-cresol on filter membrane. The GA-NF suspension in PBS was deposited on filter membrane (cellulose acetate membrane 0.45 µm) and air was pressed through the filter to remove PBS. Subsequently, the mixture containing different concentration of m-cresol, 4 mM 4-AAP and H 2 O 2 was injected to filter