Green synthesis of manganese oxide nanoparticles for the electrochemical sensing of p-nitrophenol
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Manganese oxide (MnO) NPs are widely used in contaminant sensing, drug delivery, data storage, catalysis and biomedical imaging. Green synthesis of NPs is important due to increased concern of environmental pollution. Green chemistry based synthesis of NPs is preferred due to its ecofriendly nature. In this study, MnO NPs of different sizes were synthesized in aqueous medium using clove, i.e., Syzygium aromaticum extract (CE) as reducing and stabilizing agents. These NPs were used for the electrochemical sensing of p-nitrophenol (PNP). The synthesis of MnO NPs was over in 30 min. MnO NPs of different sizes were obtained by varying metal ion concentration, metal ion volume ratio, CE concentration, CE volume ratio, and incubation temperature. Selectively, ~4 nm MnO NPs were used for electrochemical sensing of paranitrophenol. The MnO NPs modified gold electrodes detected PNP with good sensitivity, 0.16 µA µM−1 cm2. The limit of PNP detection was 15.65 µM. The MnO NPs prepared using CE based green chemistry approach is useful for PNP sensing. These NPs can also be useful for various in vivo applications in which the NPs come in human contact.
KeywordsManganese oxide nanoparticles p-Nitrophenol Electrochemical sensor Clove Syzygium aromaticum Plant extract
p-Nitrophenol is a toxic pollutant released from textile industry, leather industry, iron and steel manufacturing, foundries, pharmaceutical manufacturing, rubber processing, and electrical and electronic components production industry . PNP is also released from diesel exhaust particles and as a degradation product of the insecticides [2, 3]. Hence, PNP contaminates water and environment. Acute short-term inhalation and/or ingestion of PNP in humans leads to headache, drowsiness, nausea, and cyanosis, i.e., blue color in lips, ears and fingernails . PNP is also considered as non-genotoxic drug impurity. According to US Environment Protection Agency, US Food and Drug Administration draft and European Medicines Agency guidelines, non-genotoxic impurities can be used in amounts less than 4 mg day−1. But there is no exact threshold limit for PNP usage, and maximum usable limit has to be determined on case-to-case basis . Recent studies have documented toxic effect of PNP on animals. PNP has endocrine disrupting effect on Japanese quails. PNP exposure at even 10 µg kg−1 body weight caused hypothalamic pituitary gonadal toxicity. Hence, PNP can also hinder the reproductive processes of animals. Similar endocrine disrupting effect of PNP was observed in rats [2, 6, 7]. Newborn rats were found to experience more toxic effect of PNP than young rats . PNP also has toxic effect on soil microalgae and cyanobacteria .
Manganese (II) acetate tetrahydrate, butyl carbitol acetate (BCA), PNP and other chemicals used in the study were of analytical grade and were purchased from Merck and Aldrich. Dry clove (Syzygium aromaticum L.) flower buds were purchased from local grocery store.
CE preparation and synthesis of MnO NPs
2 g powdered clove was put in flask containing 50 ml of DDW and boiled for 2 min. The mixture was cooled and centrifuged at 7500 rpm for 10 min. The clear supernatant was labelled as CE and was stored at 4 °C. To prepare MnO NPs, 40 mM metal ion, i.e., the aqueous solution of manganese acetate (II) tetrahydrate was mixed with 1 ml CE. The reaction mixture was incubated at room temperature, i.e., 25 °C.
To obtain MnO NPs of different sizes, physiochemical factors namely, metal ion concentration, clove extract (CE) concentration, metal ion volume, CE volume and incubation temperature were varied. Metal ion concentration was varied from 1 to 80 mM. 1 ml of varying metal ion concentration was incubated with 1 ml CE at room temperature (Supplementary table ST1†). The CE concentration was varied from 0.25 to 1 ml (Supplementary table ST2†). The effect of metal ion volume ratio on MnO NPs quantity and size was evaluated by varying metal ion volume from 1 to 5 ml (Supplementary table ST3†). CE volume was varied from 1 to 4 ml. Different volumes of CE were used for the MnO NPs synthesis while keeping metal ion volume 1 ml (Supplementary Table S4†). To see the effect of incubation temperature, 1 ml of 40 mM metal ion was incubated with 1 ml CE at different temperatures ranging from 25 to 85 °C (Supplementary table ST5†).
Characterization of MnO NPs
Reaction mixtures were screened for MnO NPs synthesis using UV–Visible spectroscopy. Reaction mixtures were diluted and 2 ml of diluted sample was subjected to UV–Visible spectroscopic analysis (JASCO V-530 UV–Visible spectrophotometer). UV–Visible spectra of MnO NPs prepared using 1 ml 40 mM metal ion and 1 ml CE at 25 °C were recorded at fix time interval, 0 (just after mixing), 15, 30, 60 and 120 min, respectively. Similarly, MnO NPs obtained by varying physiochemical factors were characterized at 30 min of reaction using UV–Visible spectroscopy.
The MnO NPs were characterized for size using dynamic light scattering technique. After 30 min of incubation, the reaction mixtures were centrifuged at 7000 rpm for 10 min to isolate MnO NPs. The pellets were redispersed in double distilled water (DDW) and centrifuged again to purify MnO NPs. So, purified MnO NPs were diluted 10–20 times and were analyzed directly for size using dynamic light scattering technique (DLS, Malvern Nano-S90 zetasizer nanoseries). The size and morphology of the NPs was further characterized by field emission scanning electron microscope (FESEM, Hitachi, Sv8010, 15 kv) and transmission electron microscope (TEM, Hitachi, H-7500, 120 kV).
X-ray diffraction (XRD, Panalytical D/Max-2500) analysis was performed to check the size and structural properties of MnO NPs. Infrared spectra were recorded with a fourier transform infrared (FTIR, Perkin Elmer Spectrum 400) spectrometer. Thermal analysis of MnO NPs and CE was carried out using thermal gravimetric analysis (TGA, SDT Q600 thermal analysis). The analysis was carried out at 20–1000 °C in nitrogen gas atmosphere.
Fabrication of MnO NPs based electrochemical sensor
Clean gold electrode (surface area = 3.014 mm2) was polished with alumina slurry. The polished gold electrode was sonicated in DDW and dried thereafter. BCA and powdered MnO were mixed in 20: 80 ratio, respectively, to make slurry. The gold electrode was modified with MnO NPs slurry. The MnO NPs were coated on electrode surface so that MnO slurry cover entire electrode surface. The electrode was dried completely at 60 °C and used as so for electrochemical experiments. All the electrochemical sensing experiments were performed at room temperature using cyclic voltammeter (μAuto lab Type-III) with three-electrode configuration. Ag/AgCl with saturated KCl was used as a reference electrode. Pt wire was used as a counter electrode and the MnO NPs/BCA/gold electrode was used as working electrode. For all the measurements, 0.1 M phosphate buffer solution (pH 7.0) was used and all the solutions were prepared using DDW unless specified.
Electrochemical sensing of PNP
Where n is the number of electrons, C is the concentration in mole cm−3, D is the diffusion coefficient in cm2 s−1 and A is the area in cm2.
Results and discussion
UV–Visible and morphological characterization of MnO NPs
NPs have absorption characteristics in the UV–Visible region . The UV–Visible absorption intensity of NPs generally increases with an increase in NPs concentration . In the present study, MnO NPs showed characteristic absorption peak at around 260–270 nm. The color of aqueous metal ion solution is transparent. On addition of CE the color of metal ion solution changed to reddish dark brown from transparent. This change in color of metal ion solution acts as visible indicator of MnO NPs synthesis (Supplementary figure S1a and 1b†). Time study of MnO NPs reaction mixture at these reaction condition revealed that most of the MnO NPs synthesis was over in 30 min and further reaction did not led to an increase in MnO NPs synthesis (Supplementary figure S2a†). The DLS size of MnO NPs obtained at 30 min of reaction was 352 ± 11.493 nm (Supplementary figure S2b). With an increase in metal ion concentration from 1 to 10 mM, there was an increase in characteristic UV–Visible absorption intensity of MnO NPs. Further increase in metal ion concentration up to 40 mM has no effect on the UV–Visible absorption intensity (Supplementary figure S3a†). The peak intensity decreased with further increase in the metal ion up to 60 and 80 mM. This decrease in peak intensity may be due to the formation of bigger NPs as a result of aggregation of small NPs . The size of NPs did not change much from 1 mM (365.61 ± 42.12 nm) to 40 mM (352.06 ± 11.493 nm) metal ion concentration (Supplementary table ST1†). However, the size of NPs steadily increased with subsequent increase in metal ion concentration up to 80 mM (1012.53 ± 53.23 nm). Further as the UV–Visible absorption at 10-40 mM was almost the same, the amount of MnO NPs recovered by centrifugation was more in case of 40 mM metal ion. Therefore, 40 mM metal ion concentration was used for further experiments. As the CE concentration decreased from 1 ml to 0.25 ml, there was a decrease in the UV–Visible absorption intensity (Supplementary figure S3b†). The size of NPs increased with an decrease in CE concentration up to 0.25 ml (Supplementary Table ST2†). The decrease in the intensity and increase in size of MnO NPs may be due to formation of fewer amounts of bigger MnO NPs in the deficiency of reducing and capping agents . Therefore, 1: 1 volume ratio was better among other CE concentrations. However, as the metal ion volume increased from 1: 1 to 5: 1 (metal ion: CE) there was a decrease in the UV–Visible absorption intensity (Supplementary figure S3c†). This may be due to more dilution of CE and reaction mixture. Mixing metal ion and CE in appropriate concentration is necessary for the formation of stable small size NPs . The decrease in the peak intensity may be due to the reaction of inappropriate proportion of metal ion and CE. This might have led to formation of fewer and bigger NPs due to destabilization of small NPs [31, 32, 33]. The size of NPs increased with increasing metal ion ratio from 352.06 ± 11.49 nm at 1: 1 to 1435 ± 96.38 nm at 5: 1 ratio (Supplementary table S3†).
Thermal gravimetric analysis also supports the XRD interpretation that MnO were surrounded by various CE moieties (Fig. 3b and c). Two sharp peaks were observed at ~68 °C and ~272 °C. As the MnO has degradation temperature more than 1700 °C, the noticed peaks could have been due to degradation of stabilizing moieties around MnO NPs .
Clove extract acts as reducing and stabilizing agent during the MnO NPs synthesis. So, the surface of MnO NPs is surrounded by various organic stabilizing moieties. Careful interpretation of various peaks in the FTIR spectrum of MnO NPs gave an idea of stabilizing moieties (Supplementary figure S4a†). FTIR peaks around 504, 554, 758 and 827 cm−1 were due to MnO NPs [36, 37, 38, 39, 40]. However, peaks around 603 (R-CH group), 922 (–C–O bond), 1220 (CH2 group or C–O stretching), 1319, 1368 (C–O vibrations), 1475 (bending frequency methylene group), 1618 (aromatic C = C bond stretching), 1707 (C = O stretching vibrations), 2925 (–C = C bond) and 3393 (OH bond) cm−1 were observed mainly due to the presence of eugenol, caryophyllene, humulene and eugenol acetate in CE [41, 42, 43]. FTIR characterization of CE revealed similar peaks around 551, 600, 918, 1226, 1368, 1447, 1615, 1729, and 2936 cm−1 which were due to presence of these moieties (Supplementary figure S4b†). So, eugenol, caryophyllene, humulene and eugenol acetate moieties were mainly responsible for MnO NPs synthesis.
Electrochemical sensing of paranitrophenol using MnO NPs modified gold electrode
It is clear from above discussion that the irreversible reaction of PNP undergoes four electron gains, while the reversible reaction is a two-electron redox process. Our results are in agreement with previously documented studies [10, 15, 46]. PNP first undergoes irreversible reduction to form 4-(hydroxyamino) phenol. Then, it undergoes a pair of coupled redox, indicating the oxidation of 4-(hydroxyamino) phenol to 4-nitrosophenol, and its subsequent reversible reduction, respectively.
Sensitivity of the system
The sensitivity of the MnO NPs modified gold electrode system comes out to be 0.16 µA µM−1 cm2. Furthermore, the detection limit was calculated using 3σ IUPAC criteria. The detection limit of PNP using DPV technique comes out to be 15.65 µM.
Selectivity and interference studies
MnO NPs of different sizes have been successfully synthesized using CE as reducing and stabilizing agent. MnO NPs fabricated using this green chemistry approach has been effectively used for the electrochemical detection of PNP. The MnO NPs/BCA/gold electrode has shown good electro catalytic activity to PNP. Hence, the as-prepared electrochemical sensor has good sensitivity and low limit of detection for PNP. The interference and selectivity studies revealed that the present system has good selectivity for PNP in presence of interfering moieties. So this MnO NPs based electrochemical sensor is a robust and sensitive technique for PNP detection. Further, the MnO NPs can also be very useful for sensing other harmful chemicals and in various in vivo biological applications.
The authors are thankful to Central Instrumental Laboratory (CIL), Panjab University Chandigarh, India, for TEM, FESEM, and XRD measurements. VK is thankful to UGC for Dr. D. S. Kothari Postdoctoral Fellowship.
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