Application of synthesized copper nanoparticles using aqueous extract ofZiziphus mauritiana L. leaves as a colorimetric sensor for the detection of Ag +

The presented work demonstrates the preparation of copper nanoparticles (CuNPs) via aqueous leaves extract of Ziziphus mauritiana L. ( Zm ) using hydrazine as a reducing agent. Various parameters such as volume of extract, concentration of hydrazine hydrate, concentration of copper chloride, and pH of the solution were optimized to obtain Ziziphus mauritiana L. leaves extract derived copper nanoparticles ( Zm -CuNPs). Brownish red color was initial indication of the formation of Zm -CuNPs while it was confirmed by surface plasmon resonance (SPR) band at wavelength of 584 nm using ultraviolet-visible (UV-vis) spectroscopy. Synthesized Zm -CuNPs were characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray diffractometry (XRD). AFM images showed that the particle size of Zm -CuNPs was from 7 to 17 nm with an average size of 11.3 nm. Fabricated sensor ( Zm -CuNPs) were used as a colorimetric sensor for the detection of Ag + at a linear range between 0.67 × 10 -6 – 9.3 × 10 -6 with R 2 value of 0.992. For real water samples, limit of quantification (LOQ) and limit of detection (LOD) for Ag + was found to be 330 × 10 -9 and 100 × 10 -9 , respectively.

many researchers [1,14,15]. Research is based on continuous improvements. From an economic and an environmental point of view, a favorable method for the synthesis of nanoparticles may include working at room temperature, at neutral pH, and green reducing and capping materials. Generally, plants are considered as natural "chemical factories". It has been confirmed through various studies that the reduction of metals into their respective metals nanoparticles has been carried out through plant extracts containing polyphenols, terpenoids, alkaloids, sugars, proteins, and phenolic acids. A variety of plant extracts have been used for green synthesis of different metal nanoparticles such as cobalt [16], Ag [17], Au [18], Pd [19], ZnO [20], magnetites (Fe and Ni) [21][22], and Cu NPs [23][24][25][26]. Ziziphus mauritiana L. (Zm) is a fruit tree and well known for its medicinal as well as nutritional benefits [27,28]. It is commonly known as Jujube and locally known as 'Ber' . It is a tropical fruit found in many parts of the world including Pakistan, Africa and India. It belongs to the family Rhamnaceae [29]. It has multiple medical advantages like antihyperglycemic, antiinflammatory, antiplasmodial, and antimicrobial activities, as well as hemolytic anemia, sedative (tranquilliser), anxiolytic, diuretic, analgesic (pain reliever), and antioxidant properties. The leaves of Zm are are also very beneficial to human health and are eaten with catechu as an astringent. They are considered as diaphoretic and especially preferred for typhoid in children [10].
In the current study, the synthesis of CuNPs involves aqueous leaves extract of Zm and hydrazine hydrate as a reducing, as well as, an oxygen removing agent. Fabricated copper nanoparticles (CuNPs) were used as a colorimetric sensor for the detection of silver (Ag + ) at trace level in real water samples. As we know, there are the two novel aspects of the current work; it is the first time that Zm plant extract has been used for the synthesis of copper nanoparticles, and there are currently no other studies on copper nanoparticles for colorimetric sensing of Ag + .

Chemicals and reagents
In the current study, all chemicals and reagents were of analytical grade and used without any further treatment.

Instrumentation
Ultraviolet-visible (UV-vis) absorption spectra of synthesized Zm-CuNPs were recorded on Lambda 356 spectrophotometer (PerkinElmer Inc., Waltham, MA, USA) between 200-800 nm. Interaction between CuNPs and phytochemicals of plant extract was confirmed by FT-IR spectrophotometer (Nicolet 5700 of Thermo Madison, Thermo Electron Scientific Instruments Corp., Madison, WI, USA). Scanning electron microscopy (SEM JSM-6380 LV, JEOL Ltd., Tokyo, Japan) was used to analyze the structural characterization and morphology of prepared Zm-CuNPs. To confirm the size and shape of NPs, atomic force microscope (Agilent 5500, Agilent Technologies, Inc. Santa Clara, CA, USA) was used. Crystalline properties of fabricated Zm-CuNPs were confirmed by XRD (D-8, Bruker AXS GmbH, Karlsruhe, Germany). Digital camera was used to record visual colorimetric detection of Ag + by copper nanoparticles.

Preparation of plant leaves extract
Fresh leaves (25 g) of Ziziphus mauritiana were weighted and added into 100 mL volumetric flask. The solution was boiled at 100 °C for 15 min then cooled at room temperature. Whatman filter paper (No.1) was used for filtration of the extract to get a clear solution. The filtrate was stored at 4 °C for further synthesis of nanoparticles.

Synthesis protocol of copper nanoparticles
Various parameters, such as, volume of aqueous extract of Zm leaves, volume of reducing agent (1 M hydrazine solution), volume of precursor salt (0.01 M CuCl 2 .2H 2 O), and pH were optimized by UV-vis spectrometer and the data represented in the supplementary file as Figures S1-S4, respectively.
As per optimization study, 1 mL of 0.01 M CuCl 2 .2H 2 O, 0.5 mL of plant extract, and 2 mL of 1 M hydrazine hydrate were added into a 10 mL test tube and filled with Mili-Q water up to the mark at neutral pH. The solution mixture was left at room temperature until it appeared brown in color, which indicated the successful formation of Zm-CuNPs. No stirring or heating was involved in the process. UV-vis spectroscopy was used for initial confirmation of the formation and stability of the Zm-CuNPs.

Procedure for colorimetric sensing of silver ion
The colorimetric detection of silver ion was carried out at room temperature. For the development of calibration, known concentration of Ag + ranging from 0.67 to 9.3 µM prepared from silver nitrate stock solution (0.1 μM) mixed with 3 mL of biosynthesized copper nanoparticles solution. After a few minutes the solution was transferred into a 1 cm quartz cell to check colorimetric response. Spectroscopic study was conducted at spectral range from 200 to 800 nm by using Milli-Q water as reference reagent. Change in color from brown to blackish color was considered as a visual check and change in absorbance (delta absorbance) during LSPR study and was used as a quantitative response for the determination of silver in colorimetric system. Color change was observed from brown to blackish with change in absorbance from lower to higher against a blank solution for calibration. The analogous color alterations were also achieved with digital camera after reaction time and it was compared with previously reported detection of silver by other nanoparticles.

Preparations of real water samples
Different types of water samples such as tape water, surface water, and other sources from different localities were collected, filtered, and diluted to the desired volume. Various concentrations of analyte (Ag + ) were spiked from stock solution (0.1 μM) into 3 mL of Zm-CuNPs. The solutions were kept for 3-4 min at room temperature and then data recorded for each sample in triplicate with the help of UV-vis spectrophotometer.

UV-visible spectroscopy
In the last few decades, several studies reported that the optical response of metal nanoparticles (NPs) can be adjusted to control size and shape of synthesized nanoparticles [23]. Localized surface plasmon resonance (LSPR) modes of metallic nanoparticles (such as copper, silver, and gold) exist in the visible region of the electromagnetic spectrum. UV/ Vis spectroscopy was used for the optimization of different parameters such as precursor salt (CuCl 2 ), reducing agent (hydrazine hydrate), capping agent (leaves extract), and pH for the synthesis of small size CuNPs.
Effect of volume of aqueous extract of Ziziphus mauritiana leaves on λ max of CuNPs is shown in Figure S1. Different volumes ranging from 0.5 to 3 mL of Ziziphus mauritiana (Zm) leaves extract were used to obtain the optimum volume on the basis of blue shift which indicated smaller size of copper nanoparticles. The best result was achieved by using 0.5 mL of plant extract (Zm) at a wavelength of 590 nm by keeping the amount of precursor salt and reducing agent constant. CuNPs have an affinity to oxidize immediately in aqueous medium which is a major negative aspect for using these particles as colorimetric sensors. Hence, to provide an inert environment and to stabilize CuNPs, hydrazine hydrate was used as a reducing agent, which resists the oxidation of CuNPs by evolving nitrogen. Results show the change in LSPR band from 590 nm to a hypsochromic shift of 587 nm by using 1 mL of hydrazine hydrate 0.5-3 mL keeping the constant volume of Zm plant extract. The effect of the volume of 1 M hydrazine solution on λ max shift of CuNPs is shown in Figure  S2.
Effect of volume of precursor salt (0.01 M CuCl 2 ) ranging from 0.5 to 3 mL solutions is shown in Figure S3. The bathochromic shift and precipitation occurred with bigger particle size using an increased quantity of precursor salt (3 mL). This change in LSPR band may be result of an increased rate of nucleation with greater quantity of copper II ions present in solution. However, 1 mL of precursor salt was selected for further studies.
pH is an important factor for the stability of nanoparticles. Figure S4 shows pH effect on the blue shift, λ max, and shape of the peak, which is related to the size of copper nanoparticles in the pH range between 4 and 10.
Furthermore, various factors such as the size of nanoparticles, agglomeration, and nature of capping agents also play important roles in the position, shape, and size of LSPR band. Conversely, protonation/deprotonation of acidic group present in Zm-CuNPs might be followed due to change in pH of solution. Substantial changes occurr in shape and width of LSPR band as pH increases from 4 to 10 and pH 7 was selected as optimum pH for Zm-CuNPs on the basis of blue shift and shape of SPR band from broad to narrow. Figure 1 shows UV-visible spectra of synthesized CuNPs with respect to the stability. No significant change was observed in either the color, or in the wavelength of colloidal solution with passage of time. The results show that synthesized Zm-CuNPs under optimized parameters were found to be stable for upto 1 month. Therefore, fabricated Zm-CuNPs could be used as sensing probe during a wider period and could be stored at room temperature without using special storage conditions.

Fourier transform infrared spectroscopy
FTIR technique was used to observe interaction between CuNPs and biomolecules of plant extract. Figure 2a shows the FTIR spectrum of plant material and Figure 2b shows the spectrum of Ziziphus mauritiana extract capped CuNPs. Bands at 3430.4 cm -1 and 3348 cm -1 in the FTIR spectrum of the leaves extract are shifted to 3298 cm -1 in the FTIR spectrum of Zm-CuNPs. Moreover, band at 1729.1 cm -1 is due to carbonyl group present in the plant extract (Figure 2a) which has disappeared in FTIR spectrum of Zm-CuNPs (Figure 2b). There is also a band at 1618.3 cm -1 due to NH bending of amide group in both spectra. Also, by comparing the fingerprint region, a new signal at 674.5 cm -1 was observed in FTIR spectrum of Zm-CuNPs due to the presence of CuNPs, as it is not present in FTIR spectrum of leaves extract. Therefore, absence of carbonyl band of leave extract and appearance of new peak at 675.5 cm -1 in FTIR spectrum of Zm-CuNPs indicated that interaction of biomolecules of leaves extract occurred through carbonyl band with CuNPs.

Scanning electron microscopy (SEM)
Surface morphology of Zm-CuNPs was studied by SEM image of Zm-CuNPs (Figure 3). It was observed that NPs have a rough surface with a spongy, flower like shape. Greater catalytic activity may be due to roughness of surface of Zm-CuNPs with larger surface areas [30].

Atomic force microscopy (AFM)
AFM technique offers visualization and analysis of nanomaterial in three dimensions. As per AFM images (Figure 4a), Zm-CuNPs ranged between 7 and 17 nm with an average size of 11.3 nm which was calculated by ImageJ software. Figure  4b shows that size was increased after addition of silver into synthesized Zm-CuNPs up to 55 nm. Before sensing Zm-CuNPs were monodispersed and spherical in shape as shown in Figure 4a but after the addition of Ag + , the morphology and size of Zm-CuNPs were totally altered as shown in Figure 4b, may be due to the formation of alloy of Cu and Ag [31]. Figure 4c shows size distribution histogram for Zm-CuNPs on the basis of data achieved from AFM.

X-ray diffractometry (XRD)
X-ray powder diffraction (XRD) is a rapid analytical technique basically used for phase recognition of a crystalline material and can provide information on unit cell dimensions. Figure 5 indicates that the diffraction pattern of Zm-CuNPs was distinctive face center cubic (FCC) planes of CuNPs at (111),(100), and (220) with high crystalline level at 2θ angles of 31.7°, 45.3°, and 56.4° respectively. These standard planes at particular angles prove that Zm-CuNPs are crystalline in nature, verified with the JCPDS data (card no. 89-5899). XRD pattern is comparable with the already reported study [32]. 3.6. Colorimetric sensing of Ag + Figure 6a illustrates colorimetric performance of Zm-CuNPs after the addition of different concentrations of Ag + in the range between 0.6 × 10 -6 -9.3 × 10 -6 M using UV-vis spectroscopy. Calibration curve showing increase in absorbance with increasing concentration of Ag+ from 0.67-9.3 × 10 -6 M, inset shows color change with respective addition of Ag+. The color change was observed gradually from brown to blackish after each addition of Ag + . Figure 6b shows the linear plot of added Ag + concentration in µM versus ∆ absorbance. The LOD and LOQ values for Ag + were found to be 100 × 10 -9 ,and 330 × 10 -9 M, respectively. LOD was determined as (3*σ)/ slope of linear plot while LOQ was determined as (10*σ)/ slope of linear plot; σ is denoting the standard deviation of at least 3 blank runs measured in ∆ absorbance value.

Selectivity of sensor
Selectivity of colorimetric sensor for Ag+ in the presence of Zn 2+ , Ni 2+ , Pb 2+ , Ca 2+ , Mg 2+ , Na + , Cd 2+ , As 3+, and K + at the concentration of 10 µM was evaluated. The absorption intensity was examined under the same experimental conditions for other metal ions. From Figure 7, it is very clear that no substantial decrease of the absorption signal was observed in the presence of tested interfering ions. The results clearly indicate that there is no significant effect on absorbance and color change of Zm-CuNPs solution upon the addition of other tested metal ions, except silver ion which showed distinctive color change with the change in absorbance of surface plasmonic resonance (SPR) band.

Figures of merit
Comparative results of the currently developed colorimetric sensor with already reported studies [12,14,[33][34][35][36] are illustrated in Table 1. Although, AuNPs were successfully applied as sensors for the detection of Ag + , the good ranges but the use of Au salts makes these sensors highly expensive. Therefore, developed Zm-CuNPs based Ag + sensor is highly sensitive as LOD is much lower than most of the reported sensors. All the reported colorimetric sensors for the detection of Ag + are based on AuNPs. According to our best knowledge, our work is the first report of using CuNPs for Ag + detection. Moreover, Ziziphus mauritiana plant extract was used as capping agent which is easily available, cheap, and many active bio-molecules have been already reported in Ziziphus mauritiana plant extract [29]. Table 2 shows that aqueous extract of different plants has been reported for preparation of various nanoparticles [22,[37][38][39][40][41][42][43][44]. In the present study, aqueous leaves extract of Zm plant was used as a green material for the synthesis of CuNPs. A small size of Zm-CuNPs ranging from 7 to 17 nm was achieved, compared to reported studies.

Detection of Ag + from different real water samples
For practical application of synthesized plant extract based CuNPs sensor, a river water sample was collected from river Indus, Pakistan along with some tape water samples. Ag + was determined by standards addition method. Six known concentrations of Ag + (3, 4, and 5 µM in river water and 2, 7, and 9 μM in tape water) were prepared using spiking protocol. The desired volume of each standard was mixed with Zm-CuNPs solution and 3 replicate runs were recorded for each analysis. Detection of spiked Ag + in real water samples with percentage recovery is demonstrated in Table 3. The result  revealed that Ag + in real water sample was successfully detected by Zm-CuNPs as a colorimetric sensor with recovery between 96.2 and 102.5%.

Conclusion
We conclude that the present work focuses on a new strategy with a greener, cheaper, and facile way of producing highly stable Zm-CuNPs using a newer capping agent from Ziziphus mauritiana leaves extract. These synthesized nanoparticles are highly stable for up to one month at room temperature and neutral pH (7), without the need of any inert environment. This synthetic strategy is highly economical, more simple, efficient, and less time consuming. Moreover, these stable Zm-CuNPs were applied as a sensitive, selective, and economical colorimetric sensor for detection of silver ion at micro molar level concentration. The best merit of the study lies in the fact that highly stable CuNPs are due to strong capping potential of phytochemicals of plant extract with a small size (11.3 nm), compared to other reported studies.