Green Synthesis of Ceria Nanoparticles Using Azadirachta Indica Plant Extract: Characterization, Gas Sensing and Antibacterial Studies

In the present investigation we have fabricated the cerium dioxide (CeO 2 ) nanoparticles by green route. While preparing the cerium dioxide nanoparticles by co-precipitation method, Neem leaf extract mixed into the precursor of cerium. The synthesized nanoparticles of CeO 2 were used for the preparation of thick film sensor by using screen printing strategy. The fabricated CeO 2 sensor was characterized by XRD, SEM, EDS and TEM techniques. The structural characteristics investigated by x-ray diffraction technique (XRD). XRD confirms the formation of cubic lattice of CeO 2 material. The surface, texture, porosity characteristics were investigated from SEM analysis, while chemical composition of the material was analysed by EDS technique. The transmission electron microscopy (TEM) confirms the formation cubic lattice of the cerium dioxide material


Introduction
Material science is highly versatile branch which covers almost every field of science and technology. The new materials under the heading of nanotechnology are being reported by the researchers such as nanocomposites, modified semiconductors, transition metal doped materials, nanomaterial synthesis using plant extract etc. There is variety of materials reported by the researchers based on the green chemistry approach. The materials involve fabrication of nanomaterials from leaves, bark, and stem extract etc. Particularly the fabrication of silver and gold nanomaterials by using Azadirachta Indica leaf extract, Madhuca Longifolia extract etc. Some of the researchers reported the fabrication of nanomaterials by using medicinal plant extract, Impatiens balsamina, Lantana camara plant extract etc. to modify some surface characteristics of the nanomaterial. According to the researchers the plant extract fabrication of nanomaterials is able to modify many inherent properties of nanomaterials such as reduction of nanomaterials, enhanced surface rigidity, chemical and physical changes of nanomaterials etc.
Due to the unique physical and chemical properties, the rare earth elements have attracted the attention of the researchers. The term rare related to their difficult process of extraction. The rare earths, cerium is the most abundant metal found in the Earth's crust 1 (about 0.0046 wt%). In the periodic table, Cerium is the first element of the lanthanide series. In 1803, the existence of Ce in an oxide was first reported in Sweden and Germany. 2 This cerium oxide named as Ceria and it was given by Jacob Berzelius in Sweden. He named this oxide after the dwarf planet ceres, which itself means the Roman Goddess of Agriculture. 1 Uncommon chemical state of cerium dioxide is +4 recognised as ceria. It is a pale yellow/white powder that is formed by the calcination of the cerium oxalate or hydroxide. The 4f orbital of rare earths are shielded by the 5p and 4d electrons, that results in the fascinating catalytic properties. [3][4][5] Cerium exist in both +III and +IV oxidation states. 1 Thus, in bulk state it exists as CeO 2 and Ce2O 3 . CeO 2 nanomaterial contains a mix of cerium in the +III and +IV chemical states on the surface of the nanoparticle. 6 As the diameter of the nanoparticle decreases, the number of +III sites on the surface increases, that results in the loss of oxygen atoms (Oxygen vacancies). 7-8 CeO 2 has gained much attention in the field of nanotechnology due to their useful application for catalysts, fuel cells and fuel additives. Nanoceria (Cerium oxide nanoparticles) are widely used in chemical mechanical polishing, 9 solar cells, 10 fuel oxidation catalysis, 11 as catalyst or non-inert support catalyst and in the construction of the three way catalysts, and automotive exhaust treatment. 12 Due to lower toxicity, they also have been used as antitumor, 13 antioxidant, 14 anti-inflammation, 15 antibacterial. 16 in combating neurodegenerative diseases and as immunosnsors. 17 Recently, there are many reviews are published on the cerium and cerium oxides focusing on the synthesis, 18 defect engineering, 19 catalysis, 20 pharmaceutical properties, 21 biological and biomedical effects. 22 There are many toxic and fatal effects have been reported so far due to gas leakage and more concentration of toxic gas vapors like carbon monoxide, nitrogen dioxide, carbon dioxide etc. The reagent such as toluene is toxic to its threshold limit and hence the concentration of these gas vapors must be regulated through efficient gas sensors. The metal oxide based gas sensors are more efficient, easy to fabricate, low cost and easy to operate hence most of the researchers are diverted their attention to develop good, stable and reproducible sensors that can be operated at low temperature and are able to sense the toxic gases at lower concentration. [23][24][25][26] The present study deals with synthesis of CeO 2 nanoparticles by using Azadirachta Indica (Neem) leaf extract. The method belongs to the green synthesis method, which is low cost, facile and non-toxic method to fabricate the ceria nanoparticles. The fabricated green ceria nanoparticles were characterized by essential nano characterization techniques. Further, the prepared ceria nanoparticles were utilized as gas sensor material for some toxic gases such as (CO 2 ), (NO 2 ), C 6 H 5 -CH 3 , and petrol vapors (PV) etc. Additionally the material was utilized for antibacterial application of several pathogenic organisms.

Materials and Methods
Precursors used in the green fabrication of CeO 2 nanoparticles were of analytical grade reagents and utilized without further purification. The chemicals used were, Cerric ammonium nitrate (CAN), ethylene diaamine tetra acetic acid (EDTA), ammonium carbonate. Azadirachta Indica Neem (AIN) dried leaves were taken from local trees of nearby region of Nashik.

F a b r i c a t i o n o f C e o 2 ( C e r i u m D i o x i d e ) Nanoparticles by using Azadirachta Indica Neem (Ain) leaf extract
For fabrication of cerium dioxide nanoparticles initially the solution of Cerric ammonium nitrate (CAN) was prepared with 0.01 M in 70 cm 3 water. EDTA solution with 0.02 M was prepared in 50 ml of water. The 0.01 M CAN solution was kept over the magnetic stirrer, the magnetic niddle was kept in the solution. The magnetic stirrer was set at 1000 rpm and 50 0 C temperature to initiate the reaction with faster rate. To this 0.01M CAN solution the previously prepared AIN leaf extract (10 ml) solution mixed. To, this above mixture solution, slowly 0.02 M EDTA solution was added. After complete addition of EDTA, the 0.1 M NH 4 CO 3 solution was mixed. The NH 4 CO 3 solution was added to control the size of nanoparticles formed and prevents the reverse reaction. This overall mixture was continuously stirred for two hours. Then, the pale yellow precipitate was obtained; filter through whatmann filter paper 41. The obtained precipitates were then dried in hot air oven at 70 0 C for 30 minutes. The obtained precipitates were fired in muffle furnace at 550 0 C for 180 minutes; the faint yellow coloured CeO 2 nanoparticles were obtained. [27][28][29][30][31][32] The formation of CeO 2 chemical reaction is as depicted in scheme-1

Scheme-1 Synthesis of Cerium oxide nanoparticles Azadirachta Indica Neem (AIN) leaf extracts development
The AIN leaf extract was prepared by mixing 15g of Neem leafs in electric mixer machine. The grinded Neem leafs extract was then mixed up in one litre beaker containing distilled water. The water containing Neem leafs extract was allowed to boil at 70 0 C for 30 minutes. Then, the Neem leaf extract was centrifuged in a centrifuge machine at 2000 rpm for 20 minutes so that the sediment Neem leaf extract and supernant liquid should be separated. Finally, the Neem leaf extract was filtered out. The extract was kept in refrigerator for further use.

Preparation of thick films of CeO 2 (Cerium dioxide)
The film sensor of CeO 2 material was fabricated by Neem leaf extract using standard screen printing strategy. In this method the fabricated CeO 2 nanoparticles were used to prepared thick films. The material such as ethyl cellulose (EC), butyl carbitol acetate (BCA) used to design the film sensors considered as an organic part employed as binders. This inorganic to organic molecule ratio was kept as 70:30 respectively. Inorganic material CeO 2 was added in mechanical mixer (700 mg) and grinded half hour. Then pinch of EC was added in mortar pestle containing CeO 2 nanoparticles.
Then BCA was added drop wise to the grinded CeO 2 nanoparticles. The whole composite was stirred in Mortar pestle till the thixotropic phase (gel type) was achieved. This paste was pasted over the glass substrate, by means of photolithography technique. The screen surface made from organic polymer nylon with 42 s, size 356 was utilized for designing CeO 2 film sensor. [33][34][35][36] After, complete coating of the films, the films were dried at room temperature and then films were fired in muffle furnace at 400 0 C.

Thickness measurement of CeO 2 film sensor
The film sensor thickness was calculated by weight difference method, using equation1. By solving equation1 as per obtained data, the thickness of the CeO 2 was 4.312 μm (4312 nm).
The thickness of the film sensor was obtained in the thick region.

Results and Discussions X-ray Diffraction study
The fabricated material CeO 2 material was characterized by XRD method. The XRD technique was utilized to get structural characteristics of the CeO 2 material. The XRD pattern of CeO 2 material is as represented in Figure 1. MoKα material was utilized to generate X-rays, having intensity of 1. The mean nanoparticle size of cerium nanoparticles calculated using equation 2 was 30.57 nm.
The XRD pattern of the fabricated material CeO 2 shows matching data with 750390 JCPDS card number.

Scanning Electron microscopy (SEM)
The surface characteristics of CeO 2 material fabricated by Azadirachta Indica (Neem) leaf extract is shown in Figure 2 a-d. The images shows the surface characteristics such as texture, porosity and material topography can be seen from these images. From SEM images a-d it can be visualise that the material CeO 2 has homogeneous surface. The different dimensions grains with varied size are visible from SEM images. From image b and d it can be seen that the surface consists of small voids or cavities or interstial spaces that exhibits that the material is porous in nature. The small cavities present over the surface of this material have many merits from chemical reactivity point of view. Since the voids are very useful for adsorption phenomenon. 37-39 The design sensor CeO 2 was utilized as gas sensor, thus, these cavities plays vital role to accommodate smaller gas moieties such that there may be maximum interaction between adsorbate gas molecules and adsorbent sensor. Additionally, the material also demonstrates that there is close agglomeration of smaller grains together which are useful for adsorption characteristics.

Energy dispersive spectroscopy (EDS) study
EDS spectrum of fabricated material CeO 2 is as depicted in Figure 3. EDS analysis of the material was carried out to get the elemental composition of material. The composition of the fabricated CeO 2 material is as depicted in embedded table in figure 3. Figure 3 represents  in the ring form shows that material belongs to polycrystalline nature. [41][42][43][44] Gas sensing study of cerium oxide (CeO 2 ) sensor The fabricated thick film sensor CeO 2 was utilized as a sensor material for some toxic gases, such as toluene vapors (C 6 H 5 -CH 3 ), CO 2 , LPG and petrol vapors. The sensing mechanism was performed with the aid of gas sensing unit represented in Figure 5. The gas response (sensitivity) of the tested gases is as depicted in Figure 6-a. The gas sensitivity was recorded for the toluene vapors (C 6 H 5 -CH 3 ), CO 2 , LPG and petrol vapors. All the above listed gases have fatal effects over environment. Hence their concentration must be regulated by developing some acute sensors. Here the resistance of the film sensor was calculated by recording change in voltage against the constant resistance.
The voltage across fixed resistance is mutually convertible and measure by means of Ohm's law (V=IR). The change in resistance with elevated temperature was recorded by digital multimeter. For each cycle calculated amount of testing vapors (ppm) was allowed to enter into glass chamber, change voltage due to interaction between gas vapors and surface of metal oxide film was recorded with multimeter. After successful gas sensing cycle, the gas concentration was removed through the temperature supplement from thermostat. [45][46][47] CeO 2 sensor was successfully employed to sense the gases above listed out of that it showed highest response to the liquefied petroleum gas (LPG) vapors. For LPG vapors 93.25 % gas response at 150 0 C was recorded. Simultaneously, the sensor showed 78.20 % gas response for petrol vapors (PV) at 200 0 C, then 70.26 % gas response for toluene vapors (TV) at 200 0 C and 52.48% gas response at 250 0 C gas response was recorded. The good gas sensing results of CeO 2 is attributed to the porous nature, moderate band gap and good surface area of the cerium dioxide sensor. The gas response with change in temperature is as depicted in Figure 6-b.
The sensor initially tested for the gases with the different gas concentration from 10 1 to 10 3 gas ppm concentration. The variation of sensitivity against the vapors concentration is given in Figure 6-a. For increasing gas concentration the response signal is found to be enhanced. But further increasing the gas concentration beyond 600 ppm the gas response signal is found to be declined, the observation suggest the saturation tendency of the sensor towards tested gases at higher concentration of the tested gases. Percent selectivity of fabricated sensor cerium dioxide computed from equation 4.
% Selectivity = (S other gas /S target gas ) * 100 ... (4) S target gas -Gas response for high responded gas S other gas -Gas response for other tested gases For effective working of the sensor, it must obey the conditions such as stability, rapid response and recovery, fast sensitivity, good accessibility for the testing gases, and its reusability performance. The fabricated sensor cerium dioxide was tested for the reusability test in four cycles with time of 40 days in four runs. The test was performed at 600 ppm gas concentration for LPG and petrol vapors (PV). The results obtained from reusability test for total 4 cycles with the overall time period 40 days shown in Table 1. From table 1 it can be observed that fabricated sensor is effective to produce the gas response with frequent utilization of the sensor with specific time interval. Here, in general observation it was observed that for every run (with time interval of days) the gas response was found to declined very slightly. The declined in the gas response at every run is due to the dwindle in surface active properties of the sensor due to frequent interaction between gas vapors and sensor material. Although the reproducibility results are very satisfactory for the tested gases LPG and petrol vapors (PV), hence the fabricated sensor is reliable to sense these gases. 48-50

Response and recovery of CeO 2 sensor for LPG and PV gases
The reliability of the tested results is can be conform from by the response and recovery given by the sensor. This is prime tool to decide the effective working of the sensor in multiple runs. This test is associated with the smooth working of the sensor at desire temperature and concentration of the tested gases. The fabricated material CeO 2 sensor was effectively utilized for this parameter at the gases LPG and PV. In this research this parameter was tested for LPG and PV gas vapors. Both these tested gases have very rapid recovery and gas sensitivity time at the fabricated CeO 2 sensor.   The response and recovery time for the tested sensor is as depicted in Table 2. The response and recovery curves for LPG and petrol vapors are as shown in Figure 7.

Antibacterial study by agar well diffusion method
The antibacterial study over the prepared material CeO 2 was performed by means of agar well diffusion method. The pathogenic microorganism suspension was gained from local microbiological laboratory. The organism such as pseudomonas, staphylococcus aureus and salmonella typhae were used in antibacterial study .The antibacterial study was performed using standard agar well diffusion method. Initially the petriplates were marked as catalyst (CeO 2 material) loading from 100 μg/ ml, 200 100 μg/ml, 300 100 μg/ml, and 400 100 μg/ml for 4 different petriplates. Agar media was uniformly speared over the plates in the form nutrient broth. The agar-agar media was spread uniformly into plates with the aid of L-shaped glass mixer. Then into different quadrants the different organism suspension spread uniformly. The 6 mm cork borer was utilized prepare the wells over the bacterial broth suspension. Three different bacterial suspension were dropped into different well (quadrant) from 100 μg/ml to 400 μg/ml CeO 2 -Azadirachta Indica (Neem) extract into 4 different plates. After that these all 4 plates were incubated at 38 0 C for one day. After this incubation period, the clear zone of inhibition (ZOI) was observed around the entire well in petri plates. The ZOI was found maximum in 400 μg/ml CeO 2 -Azadirachta Indica concentrations.
The zone of inhibition around each well indicated that the material effectively worked as antibacterial agent for tested pathogenic microbes.