Surface-Enhanced Biocompatibility and Adsorption Capacity of a Zirconium Phosphate-Coated Polyaniline Composite.

The present study deals with the synthesis, characterization, and testing of a novel composite, zirconium(IV) phosphate-coated polyaniline (ZrPO4@PANI), toward the adsorption- and surface-controlled toxicity applications. Following the synthesis of the ZrPO4@PANI composite using the sol-gel route, various characterization techniques such as Fourier transform infrared spectroscopy, scanning electron microscopy, and powder X-ray diffraction were employed to confirm its surface functionality, morphology and agglomeration, and crystallinity and crystal nature, respectively. The composite was found to be effective toward the adsorptive removal of the methylene blue dye (an organic pollutant) as against the changes in the dye concentration, dose, pH, and so forth. Also, to understand the MB adsorption kinetics, the experimental data were evaluated using the Langmuir and Freundlich models and the results were described in accordance with the Langmuir isotherm model (an adsorption capacity of 120.48 mg/g at ambient temperature). In addition, the tests conducted using pseudo-first- and pseudo-second-order kinetic models confirmed the existence of pseudo-second-order rates. Furthermore, the calculation of thermodynamic parameters for the MB adsorption, namely, changes in enthalpy, entropy, and Gibbs' free energy, exhibited a spontaneous, feasible, and exothermic nature. Finally, the comparative studies of in vitro toxicity and flow cytometry confirmed that the copresence of ZrPO4 along with PANI significantly improved the biocompatibility. The outcome of the experimental results implies that the composite is capable enough of serving as the safe and low-cost adsorbent, in addition to supporting the effective capping of the surface toxicity of PANI.

Introduction

The recent increase in the usage of synthetic dyes for the product development in various industries such as paint, food, paper, plastic, textile, and so forth and at the same time the inadequate purification of industrial and household sewages lead to the water, air, soil, and ecological pollutions.[1] In general, the water color is a primary indicator of its quality, and it is a fact that even a minute amount of dye (below the 1 ppm range) in water becomes evident by its color and therefore can be considered as not suitable for drinking or maintenance of ecosystems.[2] Because of the increased pollution of synthetic dyes in the environment, the most prominent one to suffer is the aquatic system as the increased levels of dyes in water cause the inadequate passage of natural sunlight. Under such conditions, the photosynthetic metabolism of aquatic life gets strongly interrupted and cannot sustain in the presence of toxic dye constituents.[3] Among many different organic dyes responsible for environmental pollutions, methylene blue (MB) is one common industrial dye of synthetic origin, which enters the human body through water and air sources. The common effects of this dye on humans include the increased heart rate, tremors, vomiting sensation, Heinz body formation, hemoglobin depletion, methemoglobinemia, and micturition and also causes jaundice, cyanosis, quadriplegia, and so forth.[4,5] Therefore, considering the severe effects of the MB dye on humans, it is highly required to treat the MB dye-containing water before it essentially gets discharged into the environment. The various methods employed for the treating of effluents include filtration, chemical precipitation, reverse osmosis, ion exchange, electrochemical deposition, ultrafiltration, and coagulation. All these methods are different from each other in terms of efficiency of removing the dyes, cost-effectiveness, the complexity of handling the machines, need of trained personnel, and so forth. Among such methods, the adsorption technique is a more prominent and favorable one for treating the effluents because of its different features namely cost-effectiveness, simplicity in design, easy operating method, no need for manpower, and so forth. It also provides the best results without giving out any harmful degradation products and even liberates high-quality effluents.[6] In the adsorption technique, a solid support with a porous architecture is employed for the effective separation of dye molecules and the operating mechanism between the adsorbate and the adsorbent is mostly physicochemical. In a study, for example, a composite of self-assembled monolayers from chitosan, carbon nanotubes, and octa-amino polyhedral oligomeric silsesquioxanes has been developed for the isolation of Congo red and methyl orange dyes.[7] The other composites in the same category include the natural adsorbents manufactured from nanofibers containing the cellulose nanocrystals,[8] graphene oxide-polyethyleneimine hydrogels,[9] and Fe3O4/poly(allylamine)/carboxylate graphene oxide sheets formed from layer-by-layer self-assembly-mediated formation,[10] to name a few.

Polyaniline (PANI) is an excellent conductive polymer as it contains the −NH groups in addition to the aromatic unsaturation in its structure, and it is widely used in the manufacturing of printed circuit boards, antistatic coatings, and also as the corrosion protector.[11] Similarly, zirconium oxide (ZrO2) maintains some attractive features such as optical, dielectric, thermal, and chemical properties and so it is useful in many different applications to incorporate resistance and biocompatibility.[12] Taking advantage of the PANI’s cage-like structural formation capacity linked to the thermochemical resistance offered by ZrO2, the PANI/ZrO2 nanocomposite was found to be very much useful for the monolayer adsorption and its adsorption capacity toward the MB dye was found to be 77.5 mg/g.[13]

By considering the inbuilt properties offered by the PANI and zirconium derivatives, the present work is aimed to develop some non-toxic and stable adsorbents for the removal of the MB dye from the aqueous samples. The biocompatibility of the ZrPO4@PANI composite was studied by making use of an in vitro cell culture model over a 24 h period. Following the in vitro studies, the adsorption capacity of the composite for removing the MB dye was tested, where the results confirmed that the composite has lots of practical merits when used for industrial wastewater treatment. The adsorption capability of the ZrPO4@PANI composite was evaluated toward the MB removal from the aqueous media against the changes in various parameters such as adsorption capacity, optimization of different physicochemical parameters, and adsorption kinetics. Different adsorption models were employed to brief out the equilibrium isotherms and then the isotherm constants were illustrated. The thermodynamic factors, namely changes in enthalpy, entropy, and the free energy were determined to get the information on the reaction mechanism and even elaborate the scientific concept of using it traditionally.

Results and Discussion

In order to come up with the ZrPO4@PANI composite with the best possible ion-exchange capacity (IEC), we have prepared various composites using different concentrations of PANI and tabulated the IEC results in Table . Also, it was found from the results that PANI–ZrPO4 has shown superior ion exchange ability toward Na+ ions as compared to the ZrPO4 precipitate. In addition, among all the different combinations tested, the A-6 sample prepared with 0.6 M PANI provided the highest IEC value (1.98 meq/g) and so this is selected as the suitable material for carrying out the sorption studies.

Table 5

Various Combinations of the ZrPO4@PANI Composite Formed by Using Different Molar Ratios of PANI

sample	volume ratio(v/v)	Zr(IV)	PO43– (M)	aniline (M)	IEC (meq/g)
A-1	1:1:1	0.1	0.1	0.01	1.56
A-2	1:1:1	0.1	0.1	0.05	1.67
A-3	1:1:1	0.1	0.1	0.1	1.78
A-4	1:1:1	0.1	0.1	0.2	1.87
A-5	1:1:1	0.1	0.1	0.4	1.93
A-6	1:1:1	0.1	0.1	0.6	1.98
A-7	1:1:1	0.1	0.1	0.8	1.92
A-8	1:1:1	0.1	0.1	1.0	1.89
A-9	1:1:0	0.1	0.1	0	0.87

Figure provides the comparison of the scanning electron microscopy (SEM) images of (a) pure PANI, (b) ZrPO4, and (c) ZrPO4@PANI and (d) the corresponding energy-dispersive X-ray analysis (EDAX) spectrum of the ZrPO4@PANI sample. From the images, it can be observed that the rough surfaces of the ZrPO4 (Figure a) particles are becoming soft and are getting coated fully with the PANI (Figure c). In addition, a closer look of the ZrPO4@PANI’s surface (the inset of Figure c) indicates the maintenance of porous structures with several uniform pores that can provide suitable binding sites and complete localization toward the trapping of adsorbent molecules when used for the adsorption purposes. Furthermore, the EDAX spectrum in Figure d provides visual evidence for the persistence of ZnPO4 in the composite of ZrPO4@PANI, and the elements investigated are Zr, P, O, and C, thereby confirming the efficient synthesis procedure.

Figure 1

SEM analysis of (a) pure ZrPO4, (b) pure PANI, (c) ZrPO4@PANI, and (d) its EDAX spectrum.

Figure a compares the Fourier transform infrared (FTIR) spectrum of pure PANI with those of the ZrPO4@PANI composite and of the pure PANI sample, where the band observed around 3310 cm–1 is due to the NH2 stretching and the sharp peak at 2893 cm–1 is from the −CH group of the polymer chain. Similarly, the 1730 cm–1 peak is from the carbonyl bond of the carboxyl group, the 1635 cm–1 band is attributed to the NH2 bending vibration, and the 526 cm–1 band is attributed to the NH2 wagging. Also, the band located at 1552 cm–1 can be linked to the C=C stretching vibrations of the benzenoid rings, the 1410 cm–1 band is from the C–N stretching vibration, and the 1040 cm–1 band is due to the in-plane C–H bending mode.[14] However, for the ZrPO4@PANI composite, the bands around 3580 cm–1 and 3515 cm–1 indicate the asymmetric stretching of intercalated water molecules (from the splitting in the asymmetric position). The characteristic peak around 3152 cm–1 for the composite is from the symmetric stretching vibration and the peak at 1620 cm–1 is due to the deformed vibrations of O–H bonds.[15] The peak at 1252 cm–1 is linked to the out-of-plane bending vibration of P–OH and the 3455 cm–1 peak of the–NH bond stretching indicates the formation of a very strong bond. The band formed at 2954 cm–1 corresponds to C–H stretching vibrations, while the band at 1190 cm–1 corresponds to the in-plane C–H bending vibrations formed as quinoid rings doped with the PANI. Also, the bands observed at 1454 and 1520 cm–1 confirm a benzenoid and quinoid stretching mode. The bands obtained were truly coherent toward the emeraldine salt of PANI.[16]

Figure 2

(a) FTIR analysis and (b) powder XRD pattern of the ZrPO4@PANI composite; (c) comparison of the zeta potentials of pure PANI and ZrPO4@PANI at different pHs in the range of 2–11.

Comparison of the X-ray diffraction (XRD) patterns of the ZrPO4@PANI composite with that of pure PANI is shown in Figure b, where the typical reflection patterns observed for the composite are at 2θ of 11.7, 19.6, 34.2, and 37.98° corresponding to ZrPO4, while the diffraction patterns at 14.8, 20.3, 25.2, 27.4, and 29.9° are related to PANI.[17,18] However, for the pure PANI sample, the patterns are observed at 20.3°, 25.2°, and 27.4°, which indicate a highly ordered structure and crystallinity of the conducting polymer due to the continuous repetition of quinoid and benzenoid rings. Also, for the composite, the observation of two reflection patterns at 14.8 and 29.9° indicates that the PANI chain maintains the parallel and perpendicular periodicity.[19,20] The results indicate that the polymer bonding with metal phosphate does not affect the crystalline structure of PANI. Because some of the diffraction patterns of PANI coincide with the ZrPO4 patterns, all the patterns related to individual components are not seen and in addition, no impurity patterns are observed, thereby confirming the purity. The outcome of this diffractogram clarifies the formation of the ZrPO4@PANI composite in a semicrystalline phase. In addition, the mean crystallite size of the ZrPO4@PANI composite was calculated to be 35 nm using the Scherrer equation. Furthermore, the zeta potential comparison (in the pH range of 2–11) of pure PANI and the ZrPO4@PANI composite provided in Figure c indicates the changes occurring in the total potential value of the pure PANI polymer following its conjugation with ZrPO4. The observed changes include the conversion of the positive charge of PANI’s surface toward negative zeta potential values, and these slight changes may have the capacity to significantly alter the total adsorption capacity of the composite.

Influence of the Adsorbent Dose and Contact Time

The analysis of the effect of the adsorbent dosage provides information relating to the effectiveness of an adsorbent and even the capability of a dye that is to be adsorbed in the least dosage in accordance with the economic factors.[21] The dosage is considered as the main aspect among other parameters, which enhances the adsorption process by mainly limiting the adsorption capacity of the adsorbent. For studying the influence of the enhanced adsorption dosage on the percentage (%) removal ability of the dye, the adsorbent dose was selected in the range of 0.01 to 0.09 g per 100 mL. The pH was maintained at 4, the initial concentration of the dye was 50 mg/L, and the contact time was 1 h, and the results are shown in Figure a,b. The outcome illustrated in Figure a clarifies that there is an increase in the removal efficiency of the ZrPO4@PANI composite compared to the other two adsorbents of pure PANI and ZrPO4 compounds and this adsorption capacity seems to be increased with an increase in the adsorbent dose. This enhanced adsorption of the ZrPO4@PANI composite can be linked to the extra support offered by means of the composite formation and stability in the aqueous solvents. Also, the results shown in Figure b made it clear that about 95.77% removal efficiency was achieved at a dose of 0.09 g/100 mL, and it is considered as the favorable efficiency liberating inexpensive materials. The observation of such a high efficiency from the composite can be linked to the availability of more active sites for dye adsorption. The same phenomenon was also explained in terms of MB adsorption through ficus carica bast.[22] Similarly, the adsorption capacity seems to be increased with an increase in the adsorbent dosage until 0.05 g dosage. A further increase of the dosage beyond 0.05 g (per 100 mL) did not bring many changes to the % removal of the dye. As such, there was an adsorbent particles’ conglomeration that brought about an increase in the effective material’s surface area.[12,23] Therefore, 0.05 g/100 mL was recognized as the optimum dose to carry out further experiments.

Figure 3

(a) Comparison of the dye adsorption behavior of ZrPO4@PANI along with pure ZrPO4 and PANI, (b) influence of ZrPO4@PANI on dye adsorption capacity, and (c) influence of the contact time on the adsorption capacity of the ZrPO4@PANI composite.

To use any material as a potential adsorbent, understanding the effective contact time is of basic significance.[24] The equilibrium time agrees with the mechanism of diffusion control because it moves toward the surface of sorption.[25]Figure c shows the influence of the contact time on the MB removal, where about 94.68% removal occurs during the first 60 min and after a time period of 90 min, the equilibrium seems to be achieved. The adsorption rate changes due to the vacant nature of all the adsorbent sites initially, and the solute concentration gradient was also high. Later, it was observed that the vacant site of the adsorbent got decreased leading to a decrease in the adsorption rate as well as the concentration. At the end of the experiment, the decrease in the adsorption rate implies the monolayer formation, which is mainly because of the lack of active sites needed for the uptake mechanism just after maintaining the equilibrium state.[26,27]

Influence of the Dye Concentration and Solution pH

The initial concentration of the dye shows an intense influence on the removal process from the aqueous solutions. The influence of the adsorption capacity of the easy to obtain, eco-friendly, and cost-effective material was investigated with the varied dye concentrations in the range of 10 to 100 mg/100 mL. The adsorption mechanism was influensive because of the solute-solution since the adsorptive reactions are directly proportional to the solute concentration.[28] The outcome showed that the % removal tends to decrease with an increase in the concentration as shown in Figure a. It is also inferred that a very limited amount of high energy sites is present on the adsorbent’s surface. At a high dye concentration, the availability of adsorption sites tends to become less for maintaining the constant dosage of the adsorbent. As such, the removal of MB is dependent on the concentration, and the unoccupied active sites can be observed on the surface of the adsorbent at the low concentrations.[29] The interaction between the dye and adsorbent also increases with an increase in the concentration. The outcomes showed a decreased % removal with an increase in the concentration similar to that of the MB adsorption on a magnesite-halloysite composite and an increased adsorption capacity was also said to be observed for it.[30] The observation of such behavior of our ZrPO4@PANI composite can be mainly linked to the concentration gradient that serves as the main driving force along with an increase in the concentration.

Figure 4

Influence of (a) initial MB concentration and (b) solution pH on the adsorption behavior of the ZrPO4@PANI composite.

With the changes in the pH, the adsorption process is also affected to a large extent. The influence of solution pH is dependent on the number of ions present in the mixture and on the electrostatic interactions along with the adsorption surface. The solution pH affects the aqueous chemistry as well as the surface-binding sites. For the studies, the pHs in the range of 2–11 were adjusted by making use of 0.1 M HNO3 and 0.1 M NaOH solutions. Figure b shows the dye adsorption onto the surface of the ZrPO4@PANI composite, where the results indicate that there is no much change in the % removal of the dye from the solution. From the analysis, we observed only a little change, that is, a 92.67 to 95.12% increase in the % removal with an increase in the solution pH from 2 to 11, which means that the adsorption onto the ZrPO4@PANI composite is also dependent on pH. At low pHs, the adsorbent surface is positively charged and is generally suitable for ionic contaminants. Because the MB dye is cationic, the positive charges occupying the appropriate adsorption sites contended the similar positively charged dye molecules, which finally leads to a decreased dye adsorption. At high solution pHs, the negatively charged surface supports more for the adsorption of cationic dye contaminants.[12,15,31]

Adsorption Mechanism

The mechanism of removing the MB dye from the solution can occur either in an electrostatic or non-electrostatic way, where the active sites present at the adsorbent surface play a critical role. The non-electrostatic interaction involves Vander Waal’s forces, π–π stacking, and hydrophobic interactions, and the schematic representation of the interaction between MB and the adsorbent surface is shown in Scheme .[32] It is quite obvious that MB shows a planar structure comprising of many aromatic rings, which can easily form π–π stacking along with the rings on the copolymer of PANI. The electrostatic attraction among cationic MB molecules and phosphate groups plays a significant role in improving the adsorption capacity. In the process of dissolution of MB molecules, Cl̅ ionizes first and liberates out in a detached state, whereas the remaining cationic part gets adsorbed onto the surface that is negatively charged by means of electrostatic forces.

Scheme 1

Possible Mechanism of for the Removal of the MB Dye by the ZrPO4@PANI Composite

Influence of Ionic Strength

The influence of ionic strength on the dye uptake was put under study by the addition of the NaCl concentration that ranges between 0.01 to 0.5 mol/L. It was observed that with an increase in the ionic strength, there was a decrease in the MB removal capacity as shown in Figure . The observation of such a result can be due to the availability of NaCl in the solution that safeguards the electrostatic interaction between the opposing charges mainly at the solid sorbent surface and the dye molecules, leading to a decrease in the adsorbed amount with respect to an increase in the NaCl concentration.[33]

Figure 5

Influence of NaCl concentrations on the adsorption of the ZrPO4@PANI composite.

Adsorption Kinetics and Thermodynamics

The adsorption kinetic studies were carried out for estimating the contact time required for experimental adsorption to attain an equilibrium state.[34] Studies based on adsorption kinetics provide important knowledge and an understanding of the adsorption rate on the adsorbent surface and how it controls the residual time during the overall adsorption process.[35] The study of adsorption kinetics is also performed to determine the favorable model that fits well to elaborate the experimental outcome. The two kinetic models namely pseudo-first-order and pseudo-second-order were used to evaluate the time dependency of the adsorption process and also to infer the corresponding relation to the adsorption data.[36] The non-linear forms for the pseudo-first-order and pseudo-second-order are shown in eqs and 2.where qe and q denote the adsorbed dye contents at equilibrium and contact time t and k1 and k2 denote the pseudo-first-order rate constant and pseudo-second-order-rate constant, respectively.

The data were obtained for the adsorption using the pseudo-first and -second-order reactions as shown in Figure a and the outcomes are listed in Table . The value of R2 for the pseudo-second-order reaction (0.996) is more than the value of R2 for the pseudo-first-order reaction (0.986). It shows that the rate-limiting step is the chemisorption process, which includes valence force by the mechanism of either exchange or sharing of electrons.[37]

Figure 6

(a) Influence of adsorption kinetics and (b) effect of temperature on the adsorption capacity of the ZrPO4@PANI composite.

Table 1

Values of Pseudo-First and Pseudo-Second-Order Rate Constants

pseudo-first-order model			pseudo-second-order model
qe (mg/g)	k1 (1/min)	R2	qe (mg/g)	k2 (g/mg min)	R2
26.248	0.031	0.986	17.349	0.004	0.995

The thermodynamic parameters such as changes in entropy (ΔS), free energy (ΔG), and enthalpy (ΔH) were studied in order to find out the thermodynamic model of intermediate dye adsorption onto the ZrPO4@PANI composite. The parameters were estimated on the basis of equations given belowwhere Kd is the distribution coefficient, R is the universal gas constant, and T is the temperature in Kelvin.

For determining the thermodynamic parameters, the dye adsorption onto the specific material was calculated at a range of temperatures. The thermodynamic parameters were found to transfer unit mole of solution from the solution into the solid–liquid interface. The values of Kd and ΔG at certain specific temperatures were measured based on eqs and 4. Figure b shows the plot of ln(Kd) versus 1/T, where the graph is a straight line forming the slope of ΔH and the intercept of ΔS. The values of thermodynamic parameters estimated at a specific temperature range are listed in Table .

Table 2

Thermodynamic Adsorption Parameters of the Dye Onto the ZrPO4@PANI Composite at Different Temperatures

temp (K)	ΔG (kJ/mol)	ΔH (kJ/mol)	ΔS (J/mol K)
293	–1.72	35.77	127.96
298	–2.36
303	–3.00
308	–3.64
313	–4.28
318	–4.92

From the table, the negative value of ΔG specifies the spontaneous nature and feasibility of the adsorption process.[38] As the value of ΔG decreases at high temperatures, it clarifies that the higher temperature is suitable for the adsorption process. The positive values of ΔH and ΔS show that the adsorption process is an endothermic process as well as arbitrary at the adsorbent–solution interface. The accuracy of the values of ΔS and ΔH is confirmed by the unity of the R2 value from the plots. The chemisorption of ΔG was in between the range of −80 to −400 kJ/mol, whereas the physisorption of ΔG was in the range between −20 and 0 kJ/mol.[39]

Adsorption Isotherms

The adsorption isotherm explains the interaction of adsorbents and the adsorbate molecules when the whole system attains an equilibrium state. The Langmuir isotherm model is in accordance with the assumption related to the monolayer adsorption on the homogenous surface without underlying through the interaction of adsorbates and adsorbents.[40] On the other hand, the Freundlich isotherm model presumes multilayer adsorption on the heterogeneous surface.[41] Both isotherms are given in the non-linear form in eqs and 7. The theoretical model describing the experimental data was taken from the correlation coefficient.

The logarithm of both sides in the relation 7 is explained in the linear form for the Freundlich isotherm.where Ce is the equilibrium concentration of the adsorbate, qe is the amount of adsorption at the equilibrium, qm is the monolayer adsorption capacity, n is the Freundlich intensity constant, and KL and KF are the Langmuir and Freundlich constants, respectively. The values of qm and KL were calculated from the slope and the interception of straight lines of the plot 1/qe versus 1/Ce in Figure a and the estimated parameters are shown in Table . The KF and n values were determined from the linear plot of log q versus log Ce, which is shown in Figure b and the estimated parameters are shown in Table . As per the study (Table ), the Langmuir isotherm fitted well with the experimental data based on R2. The study suggests that monolayer sorption continues on the surface, which contains a limited number of adsorption sites and constant strategies required for adsorption without adsorbate transmigration on the surface. The heterogeneity factor (n) is employed to find out whether the adsorption process is linear, chemical, or physical. In the current study, Table shows that the value of the heterogeneity factor is greater than 1 showing that the adsorption of the MB dye onto the composite is a physical process.

Figure 7

(a) Langmuir and (b) Freundlich isotherm models for the MB adsorption onto the ZrPO4@PANI composite.

Table 3

Values of Langmuir and Freundlich Isotherm Model Parameters

Langmuir isotherm			Freundlich isotherm
qm (mg/g)	KL (L/mg)	R2	n	KF (mg/g)(L/mg)1/n	R2
120.48	0.010	0.999	24.378	1.02	0.987

Studies of Desorption and Reusability

The most significant aspect for reducing the cost of material is recycling the adsorbent. This study is mainly in accordance with the framework of the sustainable development concept, so it is necessary to conduct the desorption test for water rinsing. Although the use of chemical agents is considered as more effective, they are prone to generate dangerous leachates, where the released byproducts are needed to be disposed of very carefully.

The studies of desorption were carried out with 1% each of NaOH, H2SO4, and HCl. The ZrPO4@PANI composite saturated with the MB dye was kept in varied desorption media and kept in a rotatory shaker to stir regularly for 1 h at 150 rpm. Then, the adsorbent was separated and washed thoroughly with the distilled water. The results of our composite’s behavior in the presence of three different solutions are shown in Figure a. From the figure, it can be observed that HCl can be recognized as a good, favorable desorption medium as compared to H2SO4, followed by NaOH. The studies under the HCl as a desorption medium indicated that about 94% of MB dye could be desorbed in 1 h.

Figure 8

(a) Influence of desorption and (b) regeneration studies with the use of the ZrPO4@PANI composite for the MB dye.

The main advantage of the adsorbent material is its reusability. The material’s reusability at the time of MB removal from the solution is shown in Figure b. It is found that the material had about 85% MB dye removing capacity even after its simultaneous reuse five times, which confirms the stability offered by PANI as a surface-coating material in aqueous solutions. We observed an apparent decrease in the efficiency of the adsorbent per cycle, and the involvement of non-electrostatic forces limited the complete desorption process. At the end cycle, HCl was employed in the form of a desorption medium in order to completely remove the adsorbed MB ions from the surface of the ZrPO4@PANI composite.

Comparison of Adsorption Capacity with Other Available Adsorbents

The adsorption capacity of the sample was compared with those of selected other adsorbents as listed in Table . The analysis of the results provided the information that our synthesized composite is more efficient for removing the MB dye or its ions from the aqueous phase.

Table 4

Comparison of ZrPO4@PANI Composite’s Capacity for MB Adsorption with That of Other Composites

TNQCOLCOUNTTNQS. no.	adsorbent	capacity (mg/g)	refs
1	yellow passion fruit peel	6.8	(42)
2	graphene oxide	19.3	(43)
3	eucalyptus saw biochar	29.94	(44)
4	carbon nanotubes	35.4	(45)
5	ruthenium nanoparticle-loaded AC	41.60	(46)
6	Fe3O4@SiO2-EDA-COOH	43.15	(47)
7	peanut hull	68	(48)
8	reed-derived biochar	77.35	(49)
9	palm kernel fibre	95.4	(50)
10	acid-activated kaolin	101.5	(51)
11	kaolinite	102.04	(52)
12	ZrPO4@PANI	120.48	present study

Biological Significance

The biological significance of the composite formation with the ZrPO4 and PANI was studied using the in vitro cell culture systems and for that the rat liver cells were incubated with different concentrations (25–250 μg/mL) of PANI, ZrPO4, and the ZrPO4@PANI composite for a 24 h period. The results of cell viability and proliferation studies shown in Figure indicate that there is a significant reduction in the viability of cells (as compared to the untreated controls) following their exposure to the PANI samples over the 100 μg/mL concentration and this percentage loss in the viability increases gradually upon increasing the dosage up to 250 μg/mL. However, the other two samples, ZrPO4 and ZrPO4@PANI, at similar concentrations did now show much loss in the viability of cells and this can be due to the presence of ZrPO4 on the PANI surface. The significant reduction in the viability of cells following the surface loading of ZrPO4 onto the PANI polymer clearly depicts its influence on controlling the toxic responses of PANI or its intermediates. The structure of the PANI polymer maintains two phenyl rings that are conductive, which can easily form some physiologically active and/or harmful intermediates by taking advantage of the aniline or aromatic amine moieties.[53] Thus, the initiated toxic intermediates can further generate the free radical species by making use of the intracellular proteins and associated mechanisms, which all lead to cell death finally. We observed almost no effects of cell viability losses on the ZrPO4 exposed cells, meaning that they are the safer particles. In addition, the presence of these particles on the surface of PANI also enhances the biocompatibility of the PANI polymer as the losses in the cell viability values are intermediate between pure PANI and ZrPO4.

Figure 9

Comparison of the cell viability studies of PANI and ZrPO4 along with ZrPO4@PANI composite over the concentration range of 25–250 μg/mL during a 24 h period.

Following the cell viability study, the materials are tested toward their mechanism of toxicity and cell death using a flow cytometer, where the cells are stained with propidium iodide (PI) and Annexin V-FITC and the corresponding results are shown in Figures and 11, respectively. From Figure , upon staining of cells with PI as it can bind to the cell’s DNA, about 8% of the cells were found to be alive, 49% of cells to be early apoptotic, and 42% to be late apoptotic or early necrotic (Figure b). In a similar way for the ZrPO4@PANI composite-treated cells, these values get shifted to 30% of living, 63% for early apoptosis, and only 6% for late apoptosis or early necrosis (Figure d). Such an observation of a shift in these values is considered to be highly significant as the incorporation of PANI with ZrPO4 brings a number of early necrotic cells to life and early necrosis. Also, we observed that almost a similar percentage of healthy normal cells (93%; Figure c) for the ZrPO4 sample was exposed compared with that of controls (99%; Figure a), meaning that this compound does not induce any intracellular mechanisms toward the cells, thereby confirming the safe application of the particles. The same property of biocompatibility for ZrPO4 also seems to be persisted when we exposed the cells to the ZrPO4@PANI composite, that is, the availability of ZrPO4 on the surface of PANI reduced the number of dying cells by masking the highly toxic effects of the PANI polymer.

Figure 10

Comparison of apoptosis assay results for the ZrPO4@PANI composite with those of pure PANI, ZrPO4, and untreated controls.

Figure 11

Comparison of the cell apoptotic assay (Annexin-V-FITC) results: (a) no treatment, (b) treated with PANI, (c) treated with ZrPO4, and (d) treated with the ZrPO4@PANI composite over a 24 h incubation period.

Similarly, the flow cytometry results following the staining of cells with Annexin V-FITC shown in Figure confirmed again the importance of having ZrPO4 on the surface of the ZrPO4@PANI composite. From the figure, M1 and M2 correspond to the viable and apoptotically dying cell’s fluorescence intensities, where 15% of live cells of the PANI-treated cells (Figure b) increased to 87% following the exposure to the ZrPO4@PANI composite (Figure d). Also, 84% of cells experiencing the apoptotic pathway (Figure b) decreased to only 12% (Figure d), which is exclusively due to the availability of ZrPO4 on the PANI polymer. The sustainable behavior of the ZrPO4 compound can be confirmed by the observation of more than 90% live cells (Figure c), which is similar to the number of cells from the control measurements (98%; Figure a). Thus, from the adsorption and biological analysis of various samples, it can be confirmed that the presence of ZrPO4 on the PANI polymer not just improves the adsorbing capacity of PANI but also enhances the biocompatibility of PANI by protecting it as a shield from the toxic-induced responses.

Conclusions

In conclusion, we confirmed from this study that the ZrPO4@PANI composite is efficient enough for the successful removal of the MB dye from the aqueous solutions possessing optimal pHs and time intervals, where the composite shows an influensive effect on the MB adsorption process. With this composite, the adsorption equilibrium was attained in 1 h as the isothermal data were in accordance with the Langmuir and Freundlich models referring to the physical adsorption. The adsorption process was also studied using a pseudo-second-order rate kinetic model, and the composite also found to have a high reusability, that is, six consecutive desorption–adsorption cycles. In addition, the in vitro cytotoxicity studies indicated that the composite exhibits a less-toxic behavior as compared to the pure PANI polymer. The controlled toxicity behavior of PANI is supported by the copresence of ZrPO4, where the composite has enhanced biocompatibility and exhibits only the apoptosis mechanism with much of viable cells as compared to pure PANI with a direct necrotic pathway of cell death. Based on the cumulative analysis of surface-influenced adsorption capacity and non-toxicity, the ZrPO4@PANI composite has the potential to be used as a cheap, safe, and biocompatible adsorbent material for the extraction of DNA from biological samples. The additional advantage of incorporating this composite for the DNA/protein extraction applications is that ZrPO4 offers polarity and electron-rich surfaces that suit well for the effective bonding of DNA/protein groups, and effective isolation is also possible. At last, it can be stated that the ZrPO4@PANI composite is an effective and safe material that can find potential applications from the biomedical point of view considering the significant results obtained during the MB dye removal studies in an economic as well as sustainable way from the aqueous media.

Experimental Section

Materials and Methods

The chemicals used in synthesizing ZrPO4@PANI are aniline (99.98%), zirconium oxychloride (99.98%), ammonium persulphate (99.98%), and orthophosphoric acid (99.98%). All the chemicals were of analytical grade and high purity from Sigma-Aldrich (Mumbai, Maharashtra, India) and they did not need any further purification.

Synthesis of PANI

PANI in the form of a gel was synthesized by the chemical polymerization of aniline in the presence of HCl and ammonium persulphate and the detailed process is described elsewhere.[54,55] Briefly, 10% of aniline was added with 100 mL of 1 M HCl in a double-wall flask maintained at a temperature of 48 °C upon stirring continuously. Now, 150 mL of ammonium persulphate solution was added dropwise to the flask with continued stirring. After 6 h, the solution was filtered using a Buchner funnel and the leftover residue was washed thoroughly three to four times with the demineralized water (DMW). The resultant residue was dried in a desiccator and was preserved there itself until later use.

Synthesis of ZrPO4

For the synthesis of ZrPO4, a solution of 0.1 M H3PO4 and 0.1 M ZrOCl2·8H2O was taken in a beaker and was mixed thoroughly with the maintenance of pH 1 at ambient temperature. The mixing was continued for at least 60 min to obtain Zr(IV)PO4 precipitates and the precipitates were filtered and washed with ethanol at least three to four times to remove the unreacted reagents used. The white precipitate was separated by centrifugation and finally dried in an air oven at 120 overnight, and further preserved in the desiccator.

Synthesis of the ZrPO4@PANI Cation-Exchanger

The ZrPO4@PANI cation-exchanger was synthesized through a sol–gel mixing of ZrPO4 in the PANI precipitate in a 1:1 weight ratio. The material obtained as a result turned as greenish-black slurry and then it was kept for another 24 h, at ambient temperature. The gel-based on the PANI was filtered and then washed with DMW to discard the excess acids from it or the adhering material present in the trace form of ammonium persulfate. It was then dried at 60 °C in an air oven and the obtained powder is added with DMW to obtain the small-size granules, where they were converted into their acidic (H+) form by the soaking of granules in 1 mg L–1 HNO3 solution for a continuous 24 h with shaking to randomly exchange the supernatant liquid. On washing with DMW, excess acid was easily removed. It was later dried at 60 °C and then ground through a mortar and pestle to obtain the powder form of the composite. Table shows the comparison of IEC for various samples of ZrPO4@PANI formed by changing the molar ratio of aniline. The tentative structure of the composite material is shown in Scheme .

Scheme 2

Tentative Structure of the ZrPO4@PANI Composite

Instrumentation

The surface morphology was studied by scanning electron microscopy (SEM) using the FEI QUANTA 250 ESEM operated at 15.00 kV. For the elemental analysis, an EDAX detector connected to the SEM instrument was used. The powder XRD studies were performed on an XRD 6000 instrument, Shimadzu, with Cu Kα radiation λ = 1.5418 Å. For the UV–vis spectroscopic analysis, a SPECORD PLUS double beam spectrophotometer was used, and for the FTIR spectroscopy studies, a PerkinElmer FTIR instrument was used, where the samples were prepared using the KBr pellet method (wavenumber range used was 4000–400 cm–1). Also, for the zeta-potential studies, the Malvern zeta sizer 2000 & Mastersizer Micro instrument was used.

Experimental Protocol

The batch adsorption experiments were carried out in different sets containing a 250 mL adsorbent in the Erlenmeyer flask along with 100 mL MB solution comprising different initial concentrations. For that, the flasks containing the adsorbent were shaken in an isothermal water-bath shaker at a temperature of 30 ± 2 °C and 120 rpm unless it attains equilibrium. Once the decantation, as well as the filtration methods, were complete, the equilibrium dye concentration was estimated at 665 nm by making use of UV–visible spectrophotometry. The pH of the adsorption reaction was maintained using 1 N HCl and 1 N NaOH solutions. The quantity of the dye adsorbed and also the % removal of MB were measured by employing eqs and 10:where Co and Ce are the initial and equilibrium concentrations of MB in the solution (mg L–1), respectively, m is the dosage of the adsorbent (mg), and V is the volume of the MB solution (L).

Biological Assays

For testing the biocompatibility of the ZrPO4@PANI composite, the samples were tested using the in vitro BRL 3A rat liver cell culture systems toward their viability and associated cell cycle. For that, the BRL 3A cells of the rat liver origin obtained from the American type culture collection (ATCC, Manassa, VA, USA) were tested for the changes in the viability using the MTT dye (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptosis by means of Annexin V/FITC kits, where the fluorescence intensities of fluorescence isothiocyanate (FITC) and PI were measured, and the detailed experimental procedures were explained in our earlier publications.[56] Briefly, the rat liver BRL 3A cells were maintained in Eagle’s minimum essential medium (EMEM) supplemented with 10% (v/v) FBS (fetal bovine serum) and 1% antibiotics (penicillin, 100 units/mL; streptomycin, 100 μg/mL) kept at 37 °C incubators having 5% CO2 and 95% humidity.

For the MTT assay, about 104 cells/well were seeded onto 96-well plates, allowed to grow for 24 h, and when the cell growth reaches an 80% confluence level, the growth medium was replaced with fresh EMEM containing 2% (v/v) FBS and the test compounds of varying concentrations (25–250 μg/mL). The test compounds include ZrPO4@PANI and pure forms of PANI and ZrPO4, where the incubation was continued for another 24 h and following that period, the MTT reagent was added as per the manufacturer’s instruction and incubated for another 4 h. After that, the solution was replaced with 100 μL DMSO (dimethyl sulfoxide), and the formation of formazan crystals was tested by measuring the absorbance in the microplate reader at a 570 nm wavelength (BioTek Instruments, Winooski, VT, USA). The well of cells with no treatment to any external agent was selected as the control measurements and based on these controlled values, the results are calculated as the percentage decrease in the cell viability, where the values represented are the mean ± SD (standard deviation) of three replicate experiments.

To further investigate the role of ZrPO4 in controlling the surface-induced toxic mechanisms of the PANI material, the ZrPO4@PANI composite along with its precursors (ZrPO4 and PANI) have been subjected to the cell cycle analysis by making use of the flow cytometry studies.[57] It is to be noted here that the weights of materials used for the preparation of sample solutions are selected in such a way that the final amount of PANI is expected to be the same in both pure PANI and the ZrPO4@PANI composite (as the nanocomposite was formed from an equal amount of individual components). For the testing, the BRL 3A cells at a density of 2.5 × 105 cells/well in 6-well plates were seeded and when the cell growth reaches 80% confluency, the medium was replaced with a fresh medium containing 2% FBS along with the corresponding test compounds (ZrPO4@PANI, ZrPO4, and PANI) at their selected concentrations (250 μg/mL) for a 24 h incubation period. After that period, the cells were stained with PI as this agent has the capacity to intercalate the cell’s DNA and quantitatively assess the extent of replication that occurs during the cell cycle by means of the fluorescence emission that was measured.

Similarly, the apoptotic assay with the use of Annexin V-FITC was performed for investigating the influence of the ZrPO4@PANI composite on the BRL 3A liver cells. Similar to the cell cycle analysis, the apoptosis assay also involves the treatment of cells grown in the 6-well plate when the confluence levels were reached with the test compounds (ZrPO4@PANI, ZrPO4, and PANI) at selected concentrations (250 μg/mL; 24 h). After that, the cells were trypsinized and obtained the cell pellet using centrifugation at 3500 rpm (revolutions per minute) for 10 min. As per the manufacturer’s instruction, the formed pellet was dispersed in Annexin V binding V-FITC (100 μL) first and then Annexin V-FITC (5 μL) was added and incubated for another 30 min. Now, the cell cycle stage was analyzed using a flow cytometer maintained at the excitation and emission wavelengths of 490 and 670 nm, respectively, using Cell Quest Software..