Controllable phyto-synthesised copper nanoparticles for antioxidant and label-free colorimetric iron detection purposes

ABSTRACT Eco-friendly and sustainable synthesis of copper nanoparticles (CuNPs) using the aqueous leaf extract of Manilkara zapota L. and its application as a colorimetric sensor for detection of Fe(II) and Fe(III) ions is reported in this work. At first, the influence of key parameters on the formation of nanoparticles has been investigated. The optimal conditions were determined to be: pH = 11.0, the quantity of the leaf extract = 10.0 mL, copper precursor concentration = 0.5 mM, Temprature = 60 ℃, and incubation time = 30 min. The as-prepared CuNPs were characterised by various analytical techniques such as UV-Vis, TEM, XRD, XPS, and FTIR. The synthesised nanoparticles are amorphous in nature, spherical in shape with a size between 22 to 45 nm. Furthermore, the antioxidant potential of synthesised CuNPs was evaluated using Ferric-Reducing Antioxidant Power (FRAP) assay. Finally, a colorimetric method was described for the determination of Fe(II) and Fe(III) with high sensitivity and selectivity based on the CuNPs which leads to the red-shift of the absorption. Under the optimised conditions, the presented sensor showed a linear relationship over a concentration range of 10.0 μM to 270.0 μM with a LOD of 3.3 μM for both iron ions. Hence, this study has shown a great potential for the development of a cost-effective and selective colorimetric sensor utilising phytogenic CuNPs in the determination of iron ions using UV-Vis spectroscopy.


Introduction
In recent years, metal nanoparticles have gained considerable attention owing to their unique optical, electronic, magnetic, mechanical, and chemical properties, which differ greatly from bulk substances.These properties of nanoparticles attracted considerable attention for their practical applications in the areas of information storage, catalysis, photonics, electronics, photovoltaic cells, optical and biological sensors, conductive materials, coating formulations, medical, agricultural, and food [1][2][3][4][5].
The fine properties and the wide range of applications of CuNPs make their synthesis an attractive area.Several and different methods have been reported for the preparation of copper nanostructures such as vacuum vapour deposition [24] chemical reduction [25,26], thermal decomposition [27], microemulsion techniques [28], laser ablation [29], polyol method [30], sonochemical reduction [31], microwave irradiation methods [1,32], and DC arc discharge method [33].However, each of these methods suffers from different drawbacks such as employing high pressure, high energy requirement, inert atmospheres, elevated temperatures, use of toxic, hazardous, expensive reagents, and formation of side products that caused environmental pollution and restricted their use in practical and medical applications [6,8,34,35].Therefore, in this pursuit and to overcome these problems, the development of new environmentally friendly and green synthesis using non-toxic solvents and reagents with minimal wastage is highly required.Green synthesis of NPs has several advantages such as simplicity, cost-effectiveness, non-toxic, eco-friendly, low energy, time-efficient, as well as compatibility for biomedical and pharmaceutical applications [1,[36][37][38].Some reports are available for the green synthesis of copper nanoparticles using ascorbic acid [39], plant gums [40], plant extracts [35], and microorganisms [41].From these biosynthetic approaches, application of the plant extracts has many advantages such as easy to prepare, safe to handle, simple to possess wide viability of metabolites, easy to set up for large-scale production, and potent to eliminate the serious process in the preparation the cell culture and extraction [42,43].In fact, in the plant-based metal NP synthesis, plant material could act as reducing agents as well as stabilising agents.In addition to the reduction role, phytochemicals are adsorbed over the formed nanomaterial and increase their stability and also improve their surface properties.Considering that different plants contain different phytochemicals, the suggestion of different available plants for the synthesis of NPs can be help researchers and industries for the wide production of NPs.For this purpose, the synthesis of CuNPs using M. zapota leaf was done in the current work.Manilkara zapota (L.) (M.zapota) is from the Sapotaceae family and is an evergreen and glabrous tree with 8-15 m in height and with a milky juice [44,45].M. zapota is the native of Mexico and Central America and is planted in Bangladesh, India, and south of Iran, which is commonly known as Sapota or Chikoo in Hindi and Iran [45,46].The M. zapota leaves have been applied to treat a cough, cold, and diarrhoea as well as antimicrobial and antioxidant activities [47].The major compounds which have been isolated from M. zapota are lupeol acetate, apigenin-7-O-α-L-rhamnoside, oleanolic acid, myricetin-3-O-α-L-rhamnoside, tannins, caffeic acid, glycosides, and other phenolics [47][48][49][50].Some of these compounds may act as a probable reductant or stabiliser for the synthesis of nanoparticles.Application of the leaf extract of the M. zapota has been reported in the preparation of AgNPs as well [44,45].Very recently, the synthesis of CuNPs with M. zapota leaf extract was reported at a high temperature (100°C) and the effect of influencing factors on the phyto-synthesis of CuNPs has not been investigated [51].In the present work, we report the synthesis of CuNPs using M. zapota leaf extract at the moderate temperature condition (60°C) and the effect of important factors such as copper ion concentration, the quantity of M. zapota leaf extract, pH, reaction temperature, and incubation time on size and stability of particles were investigated.We investigated the in vitro antioxidant potential of these phyto-synthesised CuNPs using a ferric-reducing antioxidant (FRAP) assay.Finally, the applicability of the synthesised CuNPs as a colorimetric sensor to selective sensing of the Fe(II) and Fe(III) in aqueous solution was studied as a new application for the prepared NPs with M. zapota.Iron is the most important transition element involved in living systems, being vital to both plants and animals due to its important role in many chemical and biological processes [52].Recently, some nanomaterial-based colorimetric sensors for the determination of iron ions have been reported [53][54][55][56][57][58], however, despite their advantages, they have certain drawbacks such as preparation of the nanoparticles using a chemical method, utilisation of more expensive precursors (silver or gold salt), application of additional reagents that make these sensors costly and complicated, or utilisation of an indirect colour change resulting from a chemical reaction for the determination of iron, which is not usually a simple and reproducible task.On the other hand, some fluorescence colorimetric-sensors based on CuNPs have been suggested for iron determination with excellent performances [59][60][61] but to the best of our knowledge, this is the first research for the determination of iron ions in an aqueous solution using the phyto-synthesised CuNPs as a sensor based on UV-Vis spectroscopy.The facile preparation, excellent properties, low cost and high selectivity allow these unmodified phyto-synthesised CuNPs to be used in the colorimetric sensing of Fe(II) and Fe(III) ions.

Reagents and chemicals
Copper(II) sulphate pentahydrate (CuSO 4 .5H 2 O), ferric chloride hexahydrate (FeCl 3 .6H 2 O), ferrous sulphate heptahydrate (FeSO 4 .7H 2 O), hydrochloric acid (HCl), and sodium hydroxide (NaOH) was procured from Merck chemical company (Germany).All the utilised metal salt solutions (0.1 M) were prepared using corresponding metal chlorides or metal nitrates in deionised (DI) water.All chemicals were of reagent grade and used without any further purification.All glassware was cleaned with aqua regia and rinsed several times with deionised water.Deionised water from Millipore was employed for the reagent preparation and throughout the process.pH adjustment was conducted using dilute sodium hydroxide (NaOH) and hydrochloric acid (HCl) solutions were used wherever required.

Preparation of M. zapota leaf extract
Leaves of cultivated M. zapota were obtained from Bandar Abbas city, Hormozgan, Iran.The collected healthy leaves of M. zapota were washed completely in tap water several times.Then their washing was continued by distilled water, to overcome the dust and dirt particles.The washed leaves were then shade-dried for 2 weeks and were well-grinded using an electrical grinder.10.0 g of powdered M. zapota leaf was immersed in 250 mL of a conical flask with 150 mL of deionised water and refluxed on a magnetic stirrer at 80°C for 30 min to obtain the leaf extract.After cooling, the aqueous leaf extract was firstly filtered through normal filter paper and then by Whatman No. 1 filter paper to separate the particles and leafy materials.The obtained extract was stored at 4°C for further application in the synthesis process and was used within 1 week of preparation.Syntheis of CuNPs is shown in Scheme 1.

Application of M. zapota leaf extract in the biosynthesis of copper NPs
In a typical synthesis of copper nanoparticles, the amount of 10.0 mL of M. zapota extract was mixed with 10.0 mL of 5.0 mM aqueous solution of CuSO 4 in a 50 mL Erlenmeyer flask under vigorous stirring.After the adjustment of the pH, the reaction mixture was heated in a paraffin bath at 60°C for 2 h.The formation of CuNPs was studied by the change in colour from brown-yellowish to reddish-brown.It has been established that the CuNPs are red [8,62].
The synthesis of nanoparticles was optimised by varying the inputs parameters (keeping constant all parameters except one).To study the effect of pH, experiments were done by varying the pH (5.0, 7.0, 9.0, and 11.0) of the mixture solution, while the other factors like CuSO 4 concentration (10.0 mL, 5.0 mM), the leaf extract quantity (10.0 mL), temperature (60°C) and time (2 h) were kept constant.The effect of leaf extract quantity was investigated by adding the different volumes of C. spinosa leaf extract (5.0, 7.0, and 15.0 mL) into 10.0 mL CuSO 4 solution (5.0 mM) and pH was adjusted to 11.0 and temperature of 60°C for 2 h.The influence of copper ion concentration was studied by the addition of 10.0 mL of leaf extract to 10.0 mL Cu(II) solution with various concentrations (0.5, 1.0, 5.0, 7.0, and 10 mM).The effect of temperature was investigated by keeping the reaction solution at different temperatures (RT, 40°C, 60°C, and 80°C), while other reaction parameters were: 10.0 mL of the extract, 10.0 mL copper ion with the concentration of 0.5 mM, pH = 11.0 and 2 h incubation time at 60°C.The effect of reaction time on nanoparticle formation (at the optimum condition) was evaluated by incubation of the reaction solution at specific time intervals up to 100 min.After each experiment, the resulting CuNPs were diluted by deionised water with a ratio of 1 to 5 and analysed using a UV-Vis spectrophotometer.The colloidal dispersion of nanoparticles at optimum condition was then centrifuged at 10,000 rpm for 30 min.Then, the obtained precipitation was re-dispersed in deionised water and the centrifugation was repeated three times to remove unreacted reagents.The purified pellets were then dried and an aliquot dried powder of CuNPs was used for XRD and FTIR.

Characterisation of copper nanoparticles
UV-Vis absorption spectra were digitised using a Scinco S-3100 UV-Vis spectrophotometer (Korea) in a 1.0 cm quartz cuvette for the confirmation of CuNPs formation.A similar amount of M. zapota leaf extract, used during the synthesis of CuNPs, was applied as the blank sample during recording the UV-Vis spectra of CuNPs to clear the spectra of extract from the SPR of NPs.XRD measurements of the CuNPs synthesised by M. zapota leaf extract were carried out on the XRD Bruker D8 Advance instrument in the 2θ range of 20° to 80°.The diffraction patterns were obtained using monochromatic Cu-K α radiation (λ = 1.5406Å) with the X-Ray tube operated at 40 kV operating voltage and current of 40 mA.FTIR analysis of the dried powder CuNPs was carried out at room temperature on a Bruker alpha FT-IR spectrometer (Germany).The spectral recording was done using a diamond attenuated total reflection (ATR) accessory by scanning it in the range 600-4000 cm −1 at a resolution of 4 cm −1 .The size and morphology of the phyto-synthesised CuNPs were measured with the transmission electron microscopic examination (TEM) (Zeiss-EM10C) operated at an accelerating voltage of 80 kV on the carbon-coated grid Cu Mesh 300.X-ray photoelectron spectroscopy (XPS) experiments were performed by a Thermo Fisher Scientific K-Alpha spectrometer using Al Kα X-ray radiation.

Antioxidant activity using FRAP assay
The antioxidant abilities of M. zapota leaf extract and phyto-synthesised CuNPs were measured via ferric-reducing antioxidant power (FRAP) [63], which measures upon the reduction of ferric tripyridyltriazine (Fe 3+ -TPTZ) complex to the ferrous tripyridyltriazine (Fe 2+ -TPTZ) by our CuNPs or herbal extract (as reductant agents) at low pH, accompanied with a change in its absorbance at 593 nm.To prepare FRAP reagent, 300 mM acetate buffer (pH 3.6) was mixed with 10 mM of TPTZ solution in 40 mM HCl and 20 mM solution of FeCl 3 -6H 2 O at a 10:1:1 ratio.The prepared FRAP reagent was kept at 37°C before application.A 50 μl of different concentrations (125, 250, 500, 750, and 1000 μg/mL) of tested samples (M.zapota leaf extract and synthesised CuNPs) were permitted to react with 1.5 mL of the FRAP reagent for 10 min in the dark condition at 37°C.Then the absorbance of samples was recorded at 593 nm.To measure the antioxidant power based on FRAP assay, the initial absorbance of blank (only FRAP reagent) was recorded and was subtracted from the final absorbance of the sample (FRAP reagent added to a sample).Series of an aqueous solution of ferrous sulphate (FeSO 4 ) (0.125-2 mM) was utilised to prepare the calibration curve.All the obtained values were expressed as mM of ferrous (Fe(II)) equivalent.

Detection ability of the synthesised CuNPs
After characterising the synthesised CuNPs, it was checked for metal recognition.For such purpose, the change in the colour and subsequently in absorption spectra of the diluted CuNPs (with a ratio of 1:5) solution after the addition of each metal at the same condition (10.0 μL, 10 −1 mol L −1 into 2.5 mL CuNPs solution) was investigated by UV-Vis absorption spectra.The effect of the solution pH on the colour change was optimised by a sequence of experiments to obtain a maximum change in the colour of the assay solution and the highest sensitivity.Finally, the spectrometric detection of Fe(II) and Fe(III) was done by adding different microlitre volumes of a solution of 0.01 M Fe(II) or Fe(III) into the 2.5 mL volume of the prepared CuNP (adjusted to optimised pH).

Investigation of the ability of M. zapota leaf extract for the biosynthesis of CuNPs
Here, the aqueous extract of the leaves of M. zapota acts as a stabilising and reducing element for the phyto-synthesis of CuNPs without utilising any other extra reducing or capping compound.The main phytochemicals with hydroxyl and ketonic groups such as polyphenolic, flavonoids, terpenoids, ketones, aldehydes, and amides are responsible for the synthesis of nanoparticles, which show a chelate effect and can bind to metals [42].As it was noted previously in the introduction, the leaf extract of M. zapota contains flavonoids and polyphenolic compounds such as myricetin-3-O-α-L-rhamnoside (myricitrin), apigenin-7-O-α-L-rhamnoside, caffeic acid, tannins, glycosides, and other phenols [47][48][49][50].Therefore, these active polyphenolics in the M. zapota leaf extract show that they can act as a green part for the production of nanoparticles.
Moreover, the UV-Vis absorption band of the prepared extract as a fingerprint for the presence of phenolic nuclei, (Figure 1(a)) illustrated a significant absorption at λ max 340 nm (bond I) [37,64] belongs to the transition localised within the ring of cinnamoyl system.Also, the absorbance of π→π* transitions is related to the benzoyl ring system which appears an absorption at 274 nm (bond II) [64] and indicates the presence of phenolics.Therefore, these absorbance bands along with previous reports about the constituents of the extract [47,48] confirm the potential of the prepared extract for the biosynthesis of nanoparticles.
Furthermore, the FT-IR spectroscopy was applied to identify the possible biomolecules and phytochemical constituents in the M. zapota leaf extract which are responsible in the reduction process of CuNPs and their role as the capping compound in the biosynthesised nanoparticles.The FT-IR spectrum of the crude extract (Figure 1 peaks at 3500-3100, 2926, 1603, 1447, and 1300-1000 cm −1 which represent free OH functional groups in molecule and OH group forming hydrogen bonds, saturated hydrocarbons (C sp3 ─H), carbonyl group (C═O), stretching bond of C═C in the aromatic ring, and C─O stretching respectively.The discussed peaks are indicators for the presence of flavonoids and phenolic compounds in the M. zapota leaf extract that could be responsible for the reduction process of metal ions and construction of the metal nanoparticles [36,65].

Parameter effect on the CuNPs synthesis
The inputs parameters such as the effect of pH, the volume of leaf extract, the concentration of copper precursor, reaction temperature, and incubation time play a significant function in controlling the shape, size, dispersity, and optical features of nanoparticles.Investigation of the Surface Plasmon Resonance (SPR) is a common UV-Vis spectrophotometric tool to follow the construction of nanoparticles in the solution.The variations in bandwidth and any shifts in SPR are important illustrative features in characterising the synthesised NPs about particle shape and distribution of size [8].Hence, UV-Vis analysis were carried out to monitor and optimise shape and size distribution.

Effect of pH
Among the various parameters, the pH of the working solution is a crucial and essential parameter in the morphology, size, and stability of the nanoparticles especially for a biochemical reaction [62].Therefore, pH is the fundamental problem that we address first.The CuNPs were synthesised at different pH (5.0, 7.0, 9.0, and 11.0) and the results are presented in Fig. S1(a) in supporting information.At lower pH (pH 5.0 and 7.0) light yellowish red colour colloid was obtained, UV-Vis spectra did not show any obvious peak and spectrum includes a very broad weak band (Fig. S1(a)).The agglomeration of particles took place at this pH and the solution became turbid that its effects can be seen by increasing in the baseline.In fact, at low pH, the phyto-active compounds are protonated and their complexation with metal ions is not sufficient.Hence, in this situation, they are not considered as a good capping agent and a reducing agent.Therefore, the acidic pH suppresses the formation of the nanoparticle [66,67].While in the alkaline condition the ionisation of the phyto-phenolic compounds was accrued, the formation of CuNPs occurs rapidly and the colour of the solution immediately changed into wine-red [66].At higher pH, a large number of nanoparticles are formed and the SPR was more clear, due to the bioavailability of functional groups in the extract.At pH 11.0, dark wine red colour appeared without any turbidity, which gave a clear SPR at 510 nm.According to the discussed results, it could be expressed that the optimum pH for the preparation of CuNPs using M. zapota leaf extract was pH = 11.0.

Effect of quantity of M. zapota leaf extract
The effect of the volume of M. zapota leaf extract on the synthesis of CuNPs was investigated by exposing 5.0, 10.0 and 15.0 mL of M. zapota extract to 10.0 mL of 5.0 mM of CuSO 4 for 2 h (Fig. S1(b), supporting information).At a low quantity of leaf extract, the amount of reductant and stabiliser is low, a lesser number of Cu nuclei are formed, and the uncoated particles that form undergo uncontrolled growth and undergo aggregation or growth [68].Hence, as shown in Fig. S1(b), at 5.0 mL volume of leaf extract, a broad SPR band was obtained.With increasing the volume of leaf extract to 10.0 mL, the intensity of the SPR peak enhanced and became narrow, demonstrates a higher production of CuNPs with narrow size distribution, which is because of the availability of more reducing phyto-molecules in the leaf extract [69].With further volumes of leaf extract (15.0 mL), the broadening of SPR peak was accrued and the formed particles were unstable and agglomeration was taken place.According to the literature, an excess of reducing agents may cause instantaneous particle precipitation [70].Thus based on these results, 10.0 mL of leaf extract was considered as the optimum.

Effect of the amount of copper precursor
The effect of the amount of Cu precursor was investigated by varying the concentration of copper sulphate pentahydrate from 0.5 to 7.0 mM, while the other parameters were kept constant (leaf extract volume = 10.0 mL, pH = 11.0, and temperature = 60°C).The absorption spectra of the CuNPs formed at different concentrations of Cu 2+ ion were recorded and the results are illustrated in Fig. S1(c), supporting information.The intensity of the SPR band was enhanced as the amount of the CuSO 4 was increased from 0.5 mM to 7.0 mM.The most stable synthesised CuNPs were observed in 0.5 mM of the CuSO 4 solution that showed no agglomeration for a longer time.It is noteworthy that the possibility of agglomeration was increased with an increase in the concentration of utilised metal ions [71].Thus, 0.5 mM concentration of copper ion was chosen for further experiments.

Effect of reaction temperature
To check out the effect of temperature over the synthesis of CuNPs, 10.0 mL of the extract was added to 10.0 mL of 0.5 mM aqueous copper sulphate solution and the different reaction temperature was tested, started from room temperature and varied up to 80°C for 2 h.The effect of temperature on the formation of CuNPs is shown in Fig. S1(d) (supportinginformation).With the increase of temperature, the reduction occurred faster and the intensity of the SPR band was also enhanced.But, the precipitation of nanoparticles occurred due to the instability of the CuNPs at 80°C that increases the baseline at this temperature confirms this fact.According to the results, 60°C was selected as an optimum temperature for the reduction process.

Effect of Time
With the increase in the incubation time, the colour intensity continuously increased and looked darker.The development of the reaction between M. zapota leaf extract and metal ions to construct the CuNPs was followed by reading the absorbance values at 508 nm as a function of time (Fig. S1(e), supproting information).From Fig. S1(e) and visual observations, the aqueous solution of CuSO 4 turned to brown-reddish within 10 min which indicates the formation of CuNPs after 10 min.The SPR intensity of CuNPs increases and the maximum intense peak was detected at 30 min as shown in Fig. S1(e).The results confirmed the complete formation of CuNPs within 30 min.

Phyto-synthesis of copper nanoparticles
In this work, M. zapota leaf extract acts as reducing and stabilising components.For this goal, flavonoids and phenolics could be capped on the surface of CuNPs possibly without using any other strong binding agents and without a hazardous impact on the environment as well.The preparation of metal NPs by chemical reduction may lead to the putting of unpleased components on the surfaces of nanoparticles raising their toxicity.In fact, the reduction of copper ions into zero valent metallic nanoparticles was conducted by phenolics and flavonoids in the extract of M. zapota leaf according to the mechanism which is represented in Scheme 2. Typically, CuNPs can show absorption bands in the range of 500-600 nm.On the other hand, the colloidal solution of copper seems red due to its SPR absorption band [1,8].In the present synthesis, the prepared CuNPs indicate a single specific absorption peak at 506 nm with the red colour, which shows the formation of metallic Cu NPs.Also, no characteristic absorption band for copper oxide around 800 nm was recognised [25].

XRD analysis
The XRD pattern of the as-synthesised CuNPs in the 2θ range of 20-80° is shown in Fig. S2 (supporting information).The as-obtained CuNPs show a broad pattern, indicating the presence of amorphous copper [72] with no trace of a crystalline phase.

Morphology and size of NPs
Phyto-reduced CuNPs were characterised by TEM to identify the size and morphology of the formed nanoparticles.As revealed in Figure 2, polydisperse spherical particles and well dispersed with no aggregation.The most size of phyto-synthesised CuNPs was Scheme 2. Mechanism of the synthesised CuNPs using the aqueous extract of M. zapota.between 20 to 45 nm and was dispersed nicely.The distribution of the size of CuNPs based on TEM images is shown in a histogram in Figure 2(b).This histogram can show that the most particles are whithin the range 20-45 nm which shows a suaitable narrow rage of particles in biological activiry or sensing perpose.

Analysis using X-ray photoelectron spectroscopy (XPS)
XPS was applied to confirm the oxidation state of copper nanoparticles (Figure 3).The Cu 2p peak located at about 934.5 eV and 954.9 eV were attributed to binding energies of Cu 2p 3/2 and Cu 2p 1/2 , respectively, which proved the characteristic of CuNPs as Cu (0) [73].This result confirmed that CuNPs were successfully synthesised using M. zapota leaf extract.

FTIR spectrum
The FT-IR spectroscopy was carried out to clarify the possible phytochemicals responsible for the reduction and stabilisation of the CuNPs.The FT-IR spectrum of the synthesised CuNPs using aqueous M. zapota extract is shown in Figure 1(b).Changing the location of IR bands at 3205, 2913, 1565, 1380 and 1102 cm −1 indicate the OH functional groups, saturated hydrocarbons (C sp3 ─H), carbonyl group (C═O), stretching of C═C band in the aromatic ring, and C─OH stretching, respectively.Therefore, these results confirm that polyphenolics could be capped on the surface of formed CuNPs.The adsorption of these polyphenolics can be possibly by π-electrons interaction in the absence of other strong ligating compounds which are commonly applied for the synthesis and stabilisation of CuNPs [74].

Antioxidant activity of the phyto-synthesised CuNPs
The FRAP test provides an easy and rapid approach to check out the antioxidant activity of any component in the reaction system as reducing power.The antioxidant power of M. zapota leaf extract and synthesised CuNPs was evaluated from their capacity to reduce the TPTZ-Fe(III) complex to TPTZ-Fe(III) complex in an acidic medium.The results obtained with this method are presented in Figure 4.The obtained results depicted that the antioxidant activities of tested samples increased with the increase of concentrations of samples (dose-dependent).The FRAP values were identified in the range of 0.38 to 2.25 mM Fe(II) dependent on different concentrations (125-1000 μg/mL) of M. zapota leaf extract, demonstrates the high reducing ability of polyphenolics inside M. zapota aqueous extract.Therefore, this plant leaf extract can be used as a natural and safe reducing and capping agent and can act as a good chelator of metal ions to synthesise metallic nanoparticles.Also, the antioxidant activity of synthesised CuNPs was obtained in the range of 0.06 to 0.23 mM Fe(II), due to the presence of phytochemicals in the extract [75].

Colorimetric strategy to detect Fe(II) and Fe(III) ions
For investigating the metal recognition ability of the synthesised CuNPs, a wide range of metal ions (Li + , Na + , Mg 2+ , Ca 2+ , Bi 2+ , Al 3+ , Mn 2+ , Cu 2+ , Co 2+ , Zn 2+ , Ni 2+ , Cd 2+ , Fe 2+ , Fe 3+ , Hg 2+ , Bi 3+ , Sn 3+ and Cr 3+ ) were followed by UV-Vis absorption spectra (Figure 5).Thus, the same concentrations (4.0 × 10 −4 M) of these metals were added to the diluted solution of CuNPs. Figure 5(a,b) are the absorption spectra of CuNPs solution and corresponding colour digital images in the presence of these metal ions, respectively.The absorption spectra of CuNPs solution in the presence of metal ions are nearly the same as those of the blank solution and cause no obvious colour change.On the other hand, only Fe(II) and Fe(III) addition led to orange-brown to brown-reddish colour change, which occurred immediately after addition at room temperature.The results indicate that the sensing system exhibit favourable selectivity towards Fe(II) and Fe(III) against most of the coexisting metal cations.From the literature [76,77], carboxyl, thiol, and nitrogen-containing compounds could bind with metal ions via the metal-ligand interaction.Because the surfaces of phyto-synthesised CuNPs capped with phytochemicals that may possess -COOH, -SH 2 , and -NH 2 group, the formation of a coordination bond between these functional groups and metal ions may lead to aggregation of CuNPs and change the colour of the solution.The obtained results indicated the aggregation rate of the prepared CuNPs is better with iron ions than other metal ions.To get maximum sensitivity, the effect of pH value on the sensing system was investigated in the presence of 2.0 × 10 −4 M of Fe(II)/Fe(III), according to the peak shift of SPR band of CuNPs through changing in the sensing system.The pH of the interacting Fe(III)/Fe(II) solution was varied from 5.0 to 11.0 by increment equal to 2.0.According to Figure 6, with increasing the pH, the change in the SPR band was enhanced.To obtain a better performance, we chose pH 11.0 as the optimum pH.Despite the applied high pH, the range of iron ion concentration was not high enough to cause iron precipitation.
The UV-Vis absorption spectra of CuNPs in sensing solutions at the optimum pH with different amounts of Fe(II)/Fe(III) are recorded 2 min after each addition (Figure 7(a) and 7(b) for Fe(II) and Fe(III), respectively).A linear calibration plot was constructed with a good correlation between the Fe(II) and Fe(III) concentrations at 600 nm over the range of 10.0 μM to 270.0 μM for both iron ions with the same sensitivity (Figure 7(c,d)).Here, 10.0 μM was the value of limit of quantitation (LOQ) which has been used as the initial value of linear range.Hence, this sensor could be used for the determination of total iron in the different samples.The limit of detection (LOD), calculated as the concentration of Fe(II) or Fe(III) which produces an analytical significance, was 3.3 μM for both Fe(II) and Fe(III).To evaluate the effectiveness of the proposed method, a comparison of the method with the previously reported nanoparticles-based UV-Vis sensors for the determination of Fe 2+ and Fe 3+ ions is listed in Table 1.As is obvious, the limit of detection (LOD) and linear range of the proposed method is superior or equal to most of the existing UV-Vis Although other nanoparticles-based colorimetric sensors reported in the literature have their advantages, these methods possess one or more drawbacks such as the utilisation of hazardous chemicals, time-consuming procedure, less safe methodology, laborious or too expensive method.But, the presented method utilises unmodified synthesised CuNPs using M. zapota that hazard-free and can determine iron ions with high selectivity, sensitivity and speed and also is based on simple and available UV-Vis spectroscopy.

Conclusion
We report the green synthesis of CuNPs by the reduction of Cu 2+ ions using an aqueous extract from M. zapota leaf.The suggested methodology provides a simple, totally hazardfree, cost-effective, and environment-friendly approach.The influence of different parameters on the production of CuNPs was also studied.The optimal conditions were  determined to be: pH = 11.0, the quantity of the leaf extract = 10.0 mL, copper precursor concentration = 0.5 mM, Temprature = 60 , and incubation time = 30 min.The appearance of SPR at 508 nm reveals the formation of CuNPs.The TEM image suggests that the particles are polydisperse and mostly spherical and well dispersed.The FT-IR spectra suggest that phyto-materials could have played an important role in the stabilisation and reduction of formed nanoparticles.The electronic structures of synthesised CuNPs were Cu 0 and phytochemicals were responsible for capping ligands to the surfaces of these NPs as well as its ability the reduction of Cu 2+ .On the other hand, the FRAP assay indicated the synthesised CuNPs have antioxidant properties.Finally, a simple and costeffective colorimetric probe with high selectivity and sensitivity towards Fe(II) and Fe(III) in aqueous solutions, with a linear concentration range of 10.0 μM to 270.0 μM and LOD of 3.3 μM for both iron ions, in UV-Vis spectroscopy was proposed based on as-synthesised CuNPs without further modification, functionalization, and toxic and hazardous treatments.As iron is one of the most biologically, environmentally, and clinically important elements, the suggested method opens up the possibility of using this CuNPs based sensor for biological purposes and the research on this aspect is in progress in our laboratory.

Scheme 1 .
Scheme 1. Preparation of CuNPs using the aqueous extract of M. zapota.

Figure 2 .
Figure 2. (a) Images obtained by TEM (b) size distribution of phyto-synthesised CuNPs by aqueous leaf extract of the M. zapota.

Figure 3 .
Figure 3. XPS spectrum of the Cu regions obtained from the synthesised CuNPs.

Figure 4 .
Figure 4. Antioxidant power of the M. zapota leaf extract and synthesised CuNPs performed using FRAP assay.The represented data are mean ± SD of three independent runs each performed in duplicate.

5 .
(a) The change in the absorbance spectra of the diluted solution of the synthesised CuNPs in the presence of 4.0 × 10 −4 M of various cations; and (b) its corresponding photographic image.

Figure 6 .
Figure 6.Absorption spectra of the synthesised CuNPs without and with 2.0 × 10 −4 M concentrations of (a) Fe(II) and (b) Fe(III) solution at different pH.

Figure 7 .
Figure 7. UV-Vis spectra of synthesised CuNPs solution before and after interaction with different concentrations (a) Fe(II) and (b) Fe(III).The calibration curve of the ∆A (A 0 -A) value at 600 nm versus the concentration of (c) Fe(II) and (d) Fe(III) (Data are mean ±SD of three independent experiments).

Table 1 .
Comparison of this work and some previously reported nanoparticles-based colorimetric sensors using UV-Vis spectroscopy to detect iron ions.