Fabrication of Ag@Co-Al Layered Double Hydroxides Reinforced poly(o-phenylenediamine) Nanohybrid for Efficient Electrochemical Detection of 4-Nitrophenol, 2,4-Dinitrophenol and Uric acid at Nano Molar Level

In this paper, Co-Al layered double hydroxides (LDHs), Co-Al LDHs/poly(o-phenylenediamine) (PoPD) and Ag nanoparticles decorated Co-Al LDHs/PoPD (Ag@Co-Al LDH/PoPD) samples were prepared. The as-prepared samples were characterized by XRD, Raman, XPS, FT-IR, DRS-UV-Vis, PL and TGA techniques. The salient features of morphology and size of the samples were determined using FESEM, and HR-TEM. Then, the samples were coated on glassy carbon electrode (GCE) and employed for sensing of 4-nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP)) and uric acid (UA). It was found that Ag@Co-Al LDH/PoPD/GCE showed superior electrochemical sensing behaviour than other modified electrodes. It exhibits the detection limit (DL) of 63 nM, 50 nM and 0.28 µM for 4-NP, 2,4-DNP and UA respectively.


Results and Discussions
The crystal structure and phase purity of the samples were determined from X-ray diffraction analysis. In Fig. 3A  (pattern a), the diffraction peaks of pure Co-Al LDH are well matched with JCPDS file no. . It shows that the peaks at 11.75°, 23.62°, 34.71°, 39.36°, 46.97°, 53.24°, 60.38°, and 61.75° are corresponding to the reflection from (003), (006), (012), (104), (107), (0012), (0111) and (110) planes of the rhombohedral structured Co 6 Al 2 (OH) 16 ·4H 2 O respectively. The formation of rhombohedral phase may be due to the coordination of aluminium with the cobalt ions, as reported in literature 25 . The basal plane (003) indicates the well-organized 2D layer stacking of the prepared LDHs. There are no peaks due to oxides of cobalt and aluminium in XRD patterns of samples.
The XRD pattern of Co-Al LDH/PoPD hybrid nanostructures is shown in Fig. 3A, pattern b. It shows characteristic broad peak at 26.25° (JCPDS no: 101-1061), due to periodicity parallel chain of PoPD 26 . It denotes amorphous   Figure 3A (pattern c) predominantly shows the diffraction peaks of Ag (cubic) in Ag@Co-Al/PoPD (JCPDS file no: 901-3047). The diffraction peaks at 37.93° and 44.08° are due to crystal planes of (111) and (200) respectively 27 . The suppression of broad diffraction of peaks of PoPD might be due to dominance of more intense peaks of silver. The average crystalline size was calculated using Scherrer equation 28 , where, λ is the wavelength, K is a shape factor, θ is the diffraction angle and β is the full width of half-maximum diffraction peak. The calculated average crystallite size is ~8 nm for Ag@Co-Al LDH/PoPD. Raman spectroscopy is a powerful non-destructive technique to examine the imperfection and disordered crystal structures. The Raman spectrum of Co-Al LDHs shows the peaks at 482, 554, 1053, 1469 and 1536 cm −1 (Fig. SI). The sharp peaks at 482 and 554 cm −1 are due to stretching vibrations of metal-oxygen (M-O) bonds. The peak, 1053 cm −1 is due to stretching vibration of (CO 3 2− ), which is commonly observed for CO 3 2− intercalated LDH material 29 . The two broad peaks at 1362 and 1559 cm −1 are corresponding to the D-and G-bands, respectively. The G-band (E 2g ) assigned to stretching vibrations in the basal-plane (sp 2 domains) of PoPD polymer 30 . Further, the D-bands are usually attributed to the disorders and imperfections in the carbon crystallites. However, the Raman bands due to LDH are not observed in Ag@Co-Al LDH/PoPD. The Raman spectrum of Ag@Co-Al/PoPD (Fig. 3B, spectrum c) shows two peaks at 1355 and 1530 cm −1 that indicate the presence of PoPD. The intensity of two broad peaks due to D-and G-band is less. This is because of addition of Ag nanoparticles, which might be suppressed M-O peaks intensity 31 .
The elemental composition and their oxidation states of Ag@Co-Al LDH/PoPD were analysed by XPS. The survey spectrum and core-level spectrum of C 1 s, Co 2p and O 1 s are given in Fig. 4(a-d). Obviously, the Ag@ PoPD/Co-Al LDH showed predominant peaks due to Ag 3d, Al 2p, N 1s spectrum peaks (SI. Fig. 2a-c). The core level C 1 s spectrum (Fig. 4b) was deconvoluted into three peaks. These peaks are correspond to aromatic linked carbon (C=C, 284.9 eV), the carbonyl carbon (C=O, 287.8 eV) and the carboxylate carbon (O-C=O, 291.35 eV). Generally, the core level spectrum of Co 2p exhibits the spin-orbit split doublet. However, in our case, only a peak of Co 2p 3/2 can be observed at 781.4 eV (Fig. 4c) 32 . This result is in accordance with XRD and SAED analysis. The deconvoluted core level spectrum of O 1 s shows (    Figure 5A (spectrum a) shows the peaks at 3440, 1633, 1351, 779, 554 and 420 cm −1 . The peak in range of 3500-3250 cm −1 is related to O-H stretching vibrations adsorbed water molecule, whereas bending vibration of O-H is observed at 1633 cm −1 . The peak, 1351 cm −1 is assigned to stretching of CO 3 2− group with a double bond resonance character respectively. It should be mention that this peak is observed in all the samples 33 . The peaks at 779, 554 and 420 cm −1 are attributed to Co-O-Al, Co-O and Al-O vibrations respectively. Figure 5A (spectrum b) represents the broad peak at 3385 cm −1 that is due to N-H stretching vibration. The peaks at 1564 and 1351 cm −1 are attributed to C=N and C=C stretching mode of protonated quinoid (Q) and benzenoid (B) rings, respectively 34 . The peaks at 1107, 1061, and 969 cm −1 are assigned to CO 3 2− , C-N and C-H vibrations respectively. The weak peak observed at around 850-745 cm −1 is due to C-O-M bond and the metal-oxygen bond appeared at the lower frequency range (900-400 cm −1 ). Moreover, Fig. 5A (spectrum c) exhibits broad peaks at 3400-3500 cm −1 (ʋ (O-H)) and 1630 cm −1 (δ(H 2 O)) attributed to the intercalated water molecules. The observed peaks at 1530, 1465, 1651and 1224 cm −1 correspond to the PoPD polymer (Fig. 5B). The C-O-Co stretching vibration is observed at 763 cm −1 and the peak at 603 and 481 cm −1 may be due to Al-O and Ag-O bonds 35 .
The absorption spectra of pure Co-Al LDH, Co-Al LDH/PoPD and Ag@Co-Al LDH/PoPD nanohybrid are shown in (Fig. 5C, spectra a-c). The Co-Al LDH shows a weak peak at 515 nm, due to n-π* transition (Fig. 5C, spectrum a). The broadening of absorption range may arise due to (i) electrostatic interaction (ii) H-bonding formation between guest-host molecules and (iii) van der Waals force of attraction. However, in the case of Co-Al LDH/PoPD, three main peaks are observed at 290, 385 and 490 nm (Fig. 5C, spectrum b). The peaks at 290 and 385 nm are due to π-π* electronic transition of conjugated C=C double bond 36 . The notable blue shift is due to the quantum confinement effect that occurred during the PoPD growth.
The sharp optical absorption edges and well-defined excitonic features indicated that the synthesized Co-Al LDH particles have relatively narrow size distribution. The peak at 490 nm of the Co-Al/PoPD is slightly shifted to lower wavelength with respect to Co-Al LDH, indicating the formation of nanohybrids. Whereas, the absorption peak at 515 nm of Co-Al LDH is blue shifted to 490 nm, this shift was attributed to the strong coupling effect between Co-Al LDH and PoPD. The UV visible absorption spectrum of Ag@Co-Al LDH/PoPD is shown in Fig. 5C, spectrum (c). It shows two main peaks at 386 and 496 nm. The broad peak at 386 nm and the weak peak at 419 nm is due to π-π* transition of C=C and surface plasmon resonance of Ag respectively 37 . The weak absorption peak appears at 496 nm is due to n-π* with respect to the LDH. Figure 6(a,b) reveals that pure Co-Al LDH powder was composed of flakes like particles. Despite of the intercalated materials, high surface area with less aggregate has achieved for Co-Al LDH/ www.nature.com/scientificreports www.nature.com/scientificreports/ PoPD sample (Fig. 6c,d). The high and low magnified FESEM images of Co-Al LDH/PoPD display that Co-Al LDH flakes are spread on the PoPD matrix. This is due to the interaction of metal centres of LDH with electron rich conjugated double bonds in PoPD. Figure 6(e,f) shows the FESEM images of Ag@Co-Al LDH/PoPD sample. The images show that the Ag nanoparticles are decorated on the Co-Al /PoPD nanohybrids, because of the surface interaction between the Ag and PoPD. The even distribution of Ag on Co-Al LDH/PoPD was further confirmed by elemental mapping analysis (Fig. S4). This result is well accordance with our XRD analysis. Furthermore (SI Fig. 5a,c,e) EDS spectrum and (b, d, f) FESEM images were shown in SI Fig. S5.

Morphological features.
The surface morphology of pure Co-Al LDH, Co-Al/PoPD and Ag@ Co-Al/PoPD were analysed using HRTEM images. The low and high-magnified HRTEM images of pure Co-Al LDH are shown in Fig. 7(a,b). The Co-Al LDH adopts hexagonal flake like morphology, which clearly indicates the formation of layered structure. The interaction between Co-Al hydroxides may be due to electrostatic interaction happen during growth of LDH as reported by Duan et al. 38 . From the Fig. 7d,e, we can observe that the flakes and rod-like particles with rough surface of the Co-Al LDH layer are attached on the surface of the PoPD nanoparticles. The deposition of Co-Al LDH on PoPD can be explain by the following manner: When the o-PD monomer added to the LDH dispersion containing HCl, the o-PD monomer gets protonated and becomes positively charged, which is adsorbed on the negatively charged LDH nanoparticles due to the strong electrostatic force of attraction. After adding, ammonium persulfate as an oxidant, the oxidative polymerization reaction takes place completely and thus obtained Co-Al LDH/PoPD nanohybrids. Figure 7(g,h) shows HR-TEM images of Ag@Co-Al LDH/PoPD surface morphology at high and low magnifications. The Ag nanoparticles with average diameter of <10 nm are homogeneously In the presence of 4-NP, a pair of cathodic peaks and one anodic peak were observed. However, Co-Al LDH/GCE, Co-Al LDH/PoPD/GCE and Ag@Co-Al LDH/PoPD modified GCE show shift in cathodic peak potential for 4-NP with higher peak current and low potential than the bare GCE. The obtained reduction peak potential of 4-NP at modified GCE are as follows: Co-Al LDH [−0.04, 0.92 V], <Co-Al/PoPD [−0.14, 0.92 V] < Ag@Co-Al/PoPD [−0.08, 0.81 V]. All the corresponding data were tabulated in SI Table 1. From this, it is clear that the Ag@Co-Al LDH/PoPD/GCE has good electrochemical sensing behaviour towards the reduction of 4-NP. The reasons for enhanced sensing behaviour are due to the highly accessible active sites of the modifying layer due to nanosized dimensions of the samples as evident from HR-TEM images and excellent redox property of PoPD. Electrocatalytic sensing mechanism of 4-NP at Ag@Co-Al LDH/PoPD nanohybrids can be stated as follows: Based on the above discussion, the electrochemical redox reaction using Ag@ Co-Al LDH/PoPD in presence of 4-NP can be expressed by following stages.
Step-I: The mass transport of 4-NP from bulk solution to the electrode surface. www.nature.com/scientificreports www.nature.com/scientificreports/ Step-II: The adsorption of 4-NP on Ag@Co-Al LDH/PoPD/GCE through hydrogen bonding or electrostatic interactions and π-π interaction.
Step-III: The electron transfer reaction takes place.
Step-IV: Mass transport of product from the Ag@Co-Al LDH/PoPD/GCE surface into the bulk solution.
Based on the previous reports 39 , the redox pathway of 4-NP, which involves 6 electron process 40 , at Ag@Co-Al LDH/PoPD/GCE could be explain as follows:. In aromatic compounds (4-NP), it is known that nitro group is a well withdrawing and good leaving group, favouring electrophilic substitution reaction by the OH group present in the 4-NP at para position. The product (I) is observed at +0.23 V by the simultaneous substitution of the hydroxyl radical followed by oxidation, which is equivalent to that of the quinone and on reduction cycle, the product (II) is formed at −0.08 V and −0.81 V which is equivalent to that of hydroquinone. It indicates the further reduce the nitro groups into amino group.
pH study: Fig. 9b shows the influence of pH (3-9) on the electrochemical response of Ag@Co-Al LDH/PoPD/ GCE in 1 × 10 −3 M 4-NP. The reduction peak potential of 4-NP increases gradually with increasing pH from 3.0 to 9.0, further increase in pH, diminish the reduction peak current. At pH = 5, the reduction peak current of 4-NP is greater when compared to that in other pH values. Hence, pH = 5 was chosen for the further studies. The reason for the higher reduction peak current may be attributed to the electrostatic attraction between the 4-NP and Ag@Co-Al LDH/PoPD. Figure 9(a,b) shows the plot of pH vs I p and pH vs E p plots for Ag@Co-Al LDH/PoPD).
Scan rate effect: Electrochemical reduction of 4-NP at different scan rates was examined to know about the kinetics of the electrode reaction. The CV's of the 4-NP at Ag@Co-Al LDH/PoPD/GCE in (pH = 5) 1 × 10 −3 M 4-NP at different scan rates (20-300 mV) is shown in Fig. 8c. Figure 9c shows the plots of anodic or cathodic peak current of υ 1/2 vs. I pa (µA) for Ag@Co-Al/PoPD/GCE. The slope value was found to be greater than 0.5 which denotes that redox process of 4-NP is adsorption controlled process. The linear regression equation of the plot is given as, The relationship between ln vs. E pc (V) is shown in Fig. 9d. The obtained linear regression equations is, According to the Laviron equation, the totally reversible electrode process of 4-NP, and the relationship between the potential (E pc ) and scan rate (υ) could be expressed by the following equation 41 .
The electrode process purely depends on the modifying layer (i.e., Ag@Co-Al LDH/PoPD/GCE). The overall redox reaction of 4-NP, the number of electrons involved and obtained by following equation The number of electrons involved in this redox process is calculated as 6. Chronocoulometry: Diffusion coefficient (D) value identified from chronocoulometry for Ag@Co-Al/PoPD nanohybrids is shown in Fig. 8d. The diffusion coefficient value was determined using the Cottrell equation 42 , Differential pulse voltammetry: The differential pulse voltammograms of 4-NP at Ag@Co-Al/PoPD/GCE is displayed in Fig. 10. When increasing concentration of 4-NP, the 4-NP reduction peak current is also increased gradually and the linear concentration range is found to be 0.82 × 10 −9 to 0.17 × 10 −5 M (Ag@Co-Al LDH/PoPD). Figure 10b shows the calibration plot for 4-NP. The linear regression equation is found to be I c (µA) = −7.9629   www.nature.com/scientificreports www.nature.com/scientificreports/ when compared to bare GCE. The electrochemical sensing behaviour of Ag@Co-Al LDH/PoPD/GCE may attribute to the modifying layer that have higher surface active owing to the presence of nanoparticles as evident from HR-TEM images and the presence of -OH group that favours the formation of hydrogen bonding between 2,4-DNP and the modifying layer.
The redox pathway of 2,4-DNP at Ag@Co-Al LDH/PoPD/GCE occurs via simultaneous substitution of the hydroxyl radical by replacing eliminating two nitro groups which gives (+0.11 and +0.47 V) the product (I) 43 . The quinone further reduced as product (II) (−0.06, −0.49 and −0.72 V) and all the nitro groups are reduced to amine groups 44 .
Effect of pH: Figure 11B shows effect of pH (pH 3-9) on the electrochemical response at 1 × 10 −3 M 2,4-DNP at Ag@Co-Al LDH/PoPD modified GCE. The reduction peak current of 2,4-DNP increased when increasing pH from 3 to 9. The peak current is high at pH 5 and thus it was chosen for further electrochemical studies. In case of higher pH, the current response was less. The reason for the higher reduction current of 2,4-DNP at pH 5 may be attributes to the H-bonding interaction between nitro groups of 2,4-DNP with the modifying layer. Figure 12(a,b) shows the plot of pH vs I p and pH vs E p plots for Ag@Co-Al LDH/PoPD/GCE in 1 × 10 −3 M 2,4-DNP.
Effect of Scan rate: To study the kinetic behaviour of 2,4-DNP at Ag@Co-Al LDH/PoPD/GCE, effect of scan rates were investigated in the range of 30-300 mVs −1 (Fig. 11C). Figure 12c shows the plot of square root of scan rate versus I p , for Ag@Co-Al/PoPD/GCE. The linear regression equation of the plot is given as, From the slope value (> 0.5) the electrode surface is adsorption controlled process. The adsorption electrode process behaviour was may be attributed to the presence of two nitro groups present in 2,4-DNP, which binds effectively on the surface through H-bonding. From the relationship between ln(υ) vs E p (Fig. 12d)  Chronocoulometry studies: Diffusion coefficient (D) value was calculated by using chronocoulometry technique for the modified nanohybrids electrodes (Fig. 11D). The plot of the square root of time (t 1/2 ) against     in second step, the UA adsorbed on Ag@Co-Al LDH/PoPD/GCE. (c) Finally, adsorbed UA undergoes internal electron transfer with the formation of oxidized product of UA. Effect of pH: The pH effect on electrochemical response of 1 × 10 −3 M UA in the pH range of 3-9 is shown in Fig. 14B. The UA oxidation peak current increases gradually with increasing pH from 3 to 9, the pH 7 was chosen as the optimised pH. In case of higher pH, the current response decreased so pH 7 chosen for further analysis. However, the reason for the higher oxidation current at pH 7 attributes to the interaction of nitrogen and carbonyl   Figure 14(A,B) shows the plot of pH vs Ip and pH vs Ep plots for Ag@Co-Al LDH/PoPD/GCE in 1 × 10 −3 M UA.
Effect of scan rate: Figure 14C reveals that the oxidation peak current moved positively with increasing scan rate from 20-225 mVs −1 (pH 7). It suggests that the modified electrode has well electrochemical property and fast electron transfer ability. Further, the obtained scan rate slope is less than 0.5, so the electrode process is diffusion controlled and the number of electrons involved in UA oxidation (Eq. 4.3) is calculated as 1.91 (~2). The plots of (υ) 1/2 vs Ipa and log(υ) versus log(Epa) are shown in Fig. 15(c,d)  Chronocoulometry: Diffusion coefficient (D) was investigated using Chronocoulometry for Ag@Co-Al/ PoPD/GCE nanohybrids and shown in Fig. 14D. (inset: Fig. 14D the background subtraction, Q vs t 1/2 showed a linear relationship). By substituting A = 0.07 cm 2 , n = 2, and c = 0.001 M. The calculated diffusion coefficient for Ag@Co-Al/PoPD/GCE nanohybrids is 4.13 × 10 −12 cm 2 s −1 .
Differential pulse voltammogram of UA: The sensitivity and DL of Ag@Co-Al LDH/PoPD/GCE towards UA was further determined by DPV. Figure 16a displays the DPV's of UA oxidation at Ag@Co-Al LDH/PoPD/GCE in 0.1 M PBS. The oxidation peak current increases gradually with increasing the concentration of UA. The calibration plot for UA is shown in Fig. 16b. The linear range is found between 7.5 × 10 -7 -1.2 × 10 −5 M). The calculated DL and QL of Ag@Co-Al LDH/PoPD/GCE are 0.289 µM, and 0.9717 µM µA −1 respectively. From the experiment, it is clear that the Ag@Co-Al LDH/PoPD/GCE has relatively high sensitivity towards oxidation of UA.
Reproduceablity, stability and inference study. In order to investigate reproducibility of Ag@Co-Al/PoPD/GCE, five modified electrodes were fabricated. The response of 1 × 10 −3 M 4-NP and 2,4-DNP was measured for five modified electrodes and the relative standard deviation (RSD) was found to be 2.85%, (4-NP) and 2.80% (2,4 DNP). These RSD valuse was clearly indicates the prepared modified electrode has good reproducibility.
To find stability, Ag@Co-Al/PoPD/GCE was repeatedly used to measure 1 × 10 −3 M 4-NP, 2, 4-DNP for 20 days. The modified electrode was kept in refrigerator at 5 °C when not in use. The cathodic peak current of 4-NP and 2,4-DNP deduced to 97% and 96% respectively after seven days, further, to 89% and 86% after 20 days. The modified electrode was further studied in presence of higher concentrations of interfering species (100 fold excess of 4-NP). A 50 fold excess of K + , Na + , Mg 2+ , Ca 2+ , Cl − , SO 4 2− , and NO 3 − was not interfere in the determination of 4-NP and 2,4-DNP. However, the 25 fold excess of Zn 2+ , Cu 2+ and Fe 2+ ions interfered in the determination of 4-NP and 2,4-DNP (i.e., the reduction peak current decreases by ~5% and there is no change in the peak potential). Also, 10 fold excessive concentration of 2-nitrophenol, 3-nitrophenol, 4-chlorophenol, 2-chlorophenol interfere in the determination of 4-NP and 2,4-DNP. These results suggest that the Ag@Co-Al LDH/PoPD/GCE can be applied for real sample analysis.
In addition, effect of interfering species (dopamine) on UA determination was also studied by using Ag@Co-Al LDH/PoPD/GCE. It was found the equimolar concentration of dopamine do not interfere with UA determination.
Analytical applications. To evaluate the proposed Ag@Co-Al LDH/PoPD/GCE for the analytical applications, DPV techniuqes is used to determine the concentrations of 4-NP, 2,4-DNP and UA in real sample solutions. The standard addition method (Tables 2 and 3) was used to determine concentration of these analytes. The real water samples were collected from different places (Chennai, Trichy, Madurai and Tirunelveli) in Tamilnadu. The table  values conclude the overall results observed in the determination of 4-NP, 2,4-DNP and UA in the four independent solutions. The recoveries of 4-NP, 2,4-DNP and UA determination by Ag@Co-Al LDH/PoPD/GCE is 96.0-101.2%, 97.0-100.2% and 98.5-101.5% respectively. These results suggest that Ag@Co-Al LDH/PoPD/GCE could be use for the determination of nitroaromatics compounds and biomolecules in real samples.

conclusion
In conclusion, we have developed a simple method to synthesise Ag decorated PoPD reinforced Co-Al LDH. The resulting materials were investigated using several characterizations techniques such as XRD, Raman, FT-IR, DRS-UV vis, PL, TGA, FESEM and HR-TEM analysis. The rapid modification process, greater sensitivity and lower detection limits are the key attractive features of Ag@Co-Al LDH/PoPD/GCE. The electrochemical sensor delivered good recovery of 4-NP, 2,4-DNP and UA in different real samples. Hence, Ag@Co-Al LDH/PoPD/GCE will become great potential in the field of electrochemical sensor.  Table 3. Determination of Uric Acid.