Coenzyme based synthesis of silver nanocrystals

Enzyme and Microbial Technology 51 (2012) 231–236

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Coenzyme based synthesis of silver nanocrystals Shazia Tanvir a,∗ , Francois Oudet b , Sylviane Pulvin a , William A. Anderson c a

Laboratoire de Génie Enzymatique et Cellulaire, UMR 6022 CNRS, Université de Technologie de Compiègne, BP 20529-60205 Compiègne Cedex, France Service d’analyse Physico-Chimique, Université de Technologie de Compiègne, BP 20529-60205 Compiègne Cedex, France c Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada N2L 3G1 b

a r t i c l e

i n f o

Article history: Received 6 May 2012 Received in revised form 30 June 2012 Accepted 3 July 2012 Keywords: Silver nanoparticles Green synthesis Coenzyme as reducing agent NADPH, Stabilizer

a b s t r a c t In this work we have carried out systematic studies to identify the critical role of a coenzyme (␤-NADPH) to synthesize silver nanoparticle. Interestingly, both roles of reducing and stabilizing agents are played by ␤-NADPH. Nanoparticles obtained by this route exhibit a good crystallinity, a narrow size distribution and excellent stability in aqueous solution. The most advantageous points of this single-step environmentally friendly approach are that it takes place at nearly room temperature (20 ◦ C), overcomes many limitations encountered in other biological methods (such as the restricted concentration of AgNO3 , maintenance and manipulation of microorganisms, preparing extracts and contamination from residual reactants), bypasses the use of surfactants or capping agents and does not necessitate pH adjustment. The nano-Ag were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential, UV–vis, and energy-dispersive X-ray spectroscopy (EDX). DLS, TEM and XRD measurements showed the formation of nano-Ag with an average diameter of 20.77 ± 0.67 nm. XRD studies confirmed the nanocrystalline nature of the silver particles. Zeta potential measurements revealed that the particles are surrounded with negatively charged groups (−41 ± 5 mV) making them stable in an aqueous medium. The EDX spectrum of the silver nanoparticles confirmed the presence of elemental silver signal in high percentage. In addition to the easy and ecofriendly method of synthesis, ␤-NADPH can be regenerated by enzymatic means through glucose 6-phosphate dehydrogenase, potentially making the synthesis more cost effective. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Several physical, chemical and biological processes for synthesis of metal nanoparticles have been developed for a variety of applications [1–4]. Recently a number of studies have focused on green chemistry in the search for benign methods for the development of noble metal nanoparticles. Green synthesis of nanoparticles would have a greater industrial feasibility if the nanoparticles could be synthesized more rapidly and problems of agglomeration and polydispersity could be overcome. Silver nanoparticles are one metal of particular interest as they hold the promise to kill microbes effectively, control tumors, and can be applied in printable electronics, optoelectronic devices, and ultrasensitive chemical and biological sensors and as catalysts [4–13]. Silver nanoparticles are synthesized chemically using organic solvents and toxic reducing agents like hydrazine, sodium borohydride and N,N-dimethylformamide [14–16]. All of these are highly reactive chemicals and pose potential environmental and biological risks, especially if applied at large industrial scale. Bioinspired processes have received much

∗ Corresponding author. Tel.: +33 344234423x4075; fax: +33 344203910. E-mail address: [email protected] (S. Tanvir). 0141-0229/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2012.07.002

attention as an alternative for the production of metal nanoparticles in an environmentally friendly way, minimizing the use of toxic chemicals [17–20]. Often chemical synthesis methods lead to the presence of some toxic chemical species adsorbed on the nanoparticle surface that may have adverse effects in medical applications [21]. Indeed, immediately after the synthesis, nanoparticles may undergo surface modification, which is dependent upon the presence of reactants and adsorbing compounds. This is not an issue when synthesized with bioinspired materials as they are eco friendly and biocompatible for pharmaceutical applications [22,23]. Biosynthesis of nanoparticles is a kind of bottom up approach, where the main reaction occurring is reduction/oxidation. Though it is widely believed that the enzymes of the organisms play a major role in the bioreduction process [24,25], some studies have indicated otherwise. These studies indicated that some microorganisms could reduce silver ions where the processes of bioreduction were probably non enzymatic. For example, dried cells of Bacillus megaterium D01 and Lactobacillus sp. A09 were shown to reduce silver ions by the interaction of the silver ions with the groups on the microbial cell wall [26–28]. Metal ions are electron deficient, whereas most biological molecules (Co-enzymes, nucleotides, amino acids, vitamins,

antioxidants, proteins, etc.) are electron rich; consequently, there is a general tendency for metal ions to bind to and interact with many important biological molecules [29]. Under thermodynamically favorable reaction conditions metal ions steal electrons from biological molecules resulting into formation of nanoparticles. The reduced coenzymes such as NADPH are utilized as electron donor in many biological reactions. The redox activities of this coenzyme can be tuned by using metal ions that may promote electron transfer reactions [30]. The overall reactivity can be predicted using standard reduction potential (E◦ ) data versus standard hydrogen electrode (SHE). Since the reduction potential of the NADP+/NADPH couple is −0.32 V, theoretically the reduction of any metal having E◦ more positive than this value should be possible at room temperature, given a sufficient excess of reducing agent [31]. The standard redox potential of Ag+ /Ag is +0.80 V [32]. Thus the electrons move spontaneously toward more positive Ag+ leading to the formation of colloidal silver. The use of NADPH as a potential candidate to synthesize nanoparticle has recently been explored in aqueous solution at room temperature [33]. However, the detailed kinetic and spectroscopic work is required to probe specific interactions and underlying mechanism. Moreover, the role of the concentrations of the substrate and reducing agent has not yet been clarified with respect to the particle size and polydispersity index. In this paper, we reveal the specific role of NADPH in the reduction of silver nanoparticles through a detailed study which may be adaptable for the synthesis of other nanoparticles. NADPH is also regenerated to utilize again for the synthesis of the nano-Ag by using glucose 6-phosphate dehydrogenase. 2. Materials and methods 2.1. Chemicals and reagents ␤-Nicotinamide adenine dinucleotide 2 -phosphate reduced tetrasodium salt hydrate (NADPH) and silver nitrate were purchased from Sigma–Aldrich. NADPH regeneration system containing NADP+, glucose-6-phosphate and glucose 6phosphate dehydrogenase were obtained from Invitrogen (France). Purified water with a typical resistivity of 18 M cm was produced from a Milli-Q purification system (Millipore, Les Ulis, France). All other reagents used were analytical quality, and all aqueous solutions were prepared with Milli-Q water from a Millipore water purification system. 2.2. General Silver nitrate used as the Ag precursor and NADPH as a reducing agent were dissolved in deionized water. The effect of the reaction time, AgNO3 and NADPH concentration on nano-Ag synthesis was evaluated with UV–vis spectrophotometer. In a typical synthesis procedure, both silver nitrate and NADPH were prepared in deionized water. The effect of AgNO3 concentration was monitored in the range of 0.31–10 mM in the presence of equimolar amount of NADPH. Depending on the desired concentration of AgNO3 , the concentration of NADPH in the reaction cuvette was varied between 1 and 3 times the amount required to reduce the metal ions. During the reduction, the reaction mixture was stirred with a micro-magnetic stirring bar (PTFE coated). The reaction mixture was added in a 2 mL cuvette and was placed in a spectrophotometer equipped with a magnetic stirrer for the continuous monitoring of the evolution of the plasmon peak. The resultant nanoparticles were removed by centrifugation at 15,000 rpm for 30 min. The dispersion and centrifugation process was repeated twice to remove any unreacted silver nitrate or NADP(H) from the final product. The supernatant containing NADP+ was regenerated by glucose-6-phosphatae dehydrogenase [34]. These nanoparticles were then used for further characterizations. The transmission electron microscopy (TEM) analysis of NADPH synthesized silver nanoparticles were prepared by drop-coating synthesized silver nanoparticles solution on carboncoated copper TEM grids (40 ␮m × 40 ␮m mesh size). Samples were dried before loading them onto a specimen holder. TEM measurements were performed on a Hitachi model H 600 electron microscope operated at an accelerating voltage of 100 kV. The presence of silver particles was confirmed by energy dispersive X-ray (EDX) attached to the scanning electron microscope (FEI/Philips XL30 FEG ESEM). X-ray diffraction (INEL CPS-120 Curve Position Sensitive detector, Mn filtered FeK␣ radiation) was used to check the crystallinity of silver nanoparticles. The average particles size was calculated from the Sherrer equation [35] using the (1 1 1) reflexion of silver. X-ray diffraction experiments were conducted as follows. Drops of water-suspended Ag particles were dried on glass slides. The X-ray diffractogram

A

2.5

Absorbance (410 nm)

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2

0.31 mM 0.62 mM 1 25 mM 1.25 M 2.5 mM 5 mM 10 mM

1.5 1 0.5 0 0

5

10

20

15

Time (hours)

B

3 2.5

Absorbance

232

2

20 h

1.5 1 0.5

0

0 360

460

560

660

Wavelength (nm) Fig. 1. Reduction of silver nitrate solution into nano-Ag as a function of time at different concentrations of AgNO3 using equimolar concentration of NADPH as a reducing agent (A). Measurements were made at 20 ◦ C recorded every 30 min for 20 h after correcting the baseline for the same concentration of NADPH and AgNO3 (B) UV–vis spectra of an aqueous solution of 0.62 mM AgNO3 with the same concentration of NADPH.

was recorded with an exposition time of approximately 8 h. The contribution of the glass slide was then subtracted in order to extract the purest signal from the silver sample.

2.3. Dynamic light scattering and zeta potential measurement The hydrodynamic diameter and zeta potential of the synthesized nanoparticles was measured. Particle sizes (hydrodynamic diameters), polydispersity index, and zeta potential were measured on a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) operating with a He–Ne laser at a wavelength of 633 nm using backscattered light. The results are the means of triplicate runs, and in each run, 10 measurements were made. A refractive index (RI) of 1.5 was used, and the viscosity of the sample was assumed to be the viscosity of the dispersant [36]. The absorption value of the nano-Ag at 633 nm was measured by UV–vis spectroscopy and used as a correction factor in the experimental parameters used for light scattering. The sample was vortexed, and transferred into either 2 mL cuvettes or a 1 mL clear zeta potential cuvette (DTS1060, Malvern). The electrophoretic mobility of the sample was measured and converted into the zeta potential by applying the Henry equation. The data were collected and analyzed with the Dispersion Technology software 5.1 (Malvern) producing curves for the particles size as intensity distribution or diagrams for the zeta potential as a distribution versus total counts.

3. Results and discussion To investigate the specific roles of each reagent in this system, we started from a simple reduction reaction by simply mixing the silver source AgNO3 and the reducing agent NADPH. Under magnetic stirring, the solution changed color from light yellow to brownish in about few hours which is indicative of the formation of silver nanoparticles. The real-time measurement of the formation of nano-Ag was evaluated with a UV–vis spectrophotometer at a 30 min time interval. Without NADPH, keeping all other conditions constant did not yield any particles even after 36 h. The nano-Ag plasmon band appears near 410 nm in this work (Fig. 1). These

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Fig. 3. Effect of varying the concentration of NADPH at 0.5 mM AgNO3 on nano-Ag synthesis: (A) size distribution profile by intensity and (B) kinetics of the particle formation.

Fig. 2. (A) A representative of TEM images. (B) Size distribution by intensity from dynamic light scattering measurements indicating average hydrodynamic diameters (HD) for the different concentrations of AgNO3 .

color changes arise because of the excitation of surface plasmon vibrations with the silver nanoparticles [37]. 3.1. Particle size and its chemical composition Fig. 2A shows a representative TEM image recorded from the drop-coated film of the silver nanoparticles synthesized by treating the silver nitrate solution with NADPH. Dynamic light scattering is used by the instrument to determine the size distribution of particles by measuring dynamic fluctuations of light scattering intensity caused by the Brownian motion of the particle (Fig. 2B). This technique yields a hydrodynamic diameter that is calculated via the Stokes–Einstein equation from the mentioned measurements. The measurement gives the average hydrodynamic diameter of the particles and the polydispersity index (PDI) (Table 1). The particle size curve of nano-Ag by intensity (Fig. 2B) shows an average size of

Table 1 Effect of varying the concentration of AgNO3 on the polydispersity index (PDI) and hydrodynamic diameter (HD) of nano-Ag using the equimolar concentration of NADPH. AgNO3 :NADPH (mM, 1:1)

PDI

HD (nm)

10 5 2.5 1.25 0.62 0.31

0.178 0.173 0.156 0.118 0.105 0.122

19.99 20.25 20.40 21.43 21.65 20.95

20.77 ± 0.67 nm. The PDI scale ranges from 0 to 1, with 0 being monodisperse and 1 being polydisperse. The polydispersity index is a measure of the distribution of nanoparticles. A polydispersity index greater than 0.5 indicates possible aggregation and a broad particle size distribution. The influence of the increasing concentration of AgNO3 at same concentration of NADPH was tested on the rate of reaction and particle size. Table 1, showed a PDI of less than 0.2, indicating a narrow size distribution resulting from concentrations ranging from 0.31 to 10 mM. Figs. 3 and 4 indicate the role of NADPH. When the concentration of NADPH was increased to 3 times both rate of the reaction (Fig. 3B) and PDI (Table 2) increased suggesting the formation of large particles with aggregates, which was also confirmed by DLS (Fig. 3A) and TEM (Fig. 4A–C) characterization. When NADPH and AgNO3 were added with 1:1 molar ratio, nano-Ag with high yield and great uniformity can be obtained as confirmed by TEM images, hydrodynamic diameter and PDI. In the analysis of the silver nanoparticles by energy dispersive spectroscopy (EDS), the presence of the elemental silver signal was confirmed (Fig. 5). The Ag nanocrystallites display an optical absorption band peaking at 3 keV, which is typical of the absorption of metallic Ag nanocrystallites due to the surface plasmon resonance [38]. A typical X-ray diffractogram is presented in Fig. 6. Reflexions from the classical (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of face-centered cubic lattices are evidenced and confirm the crystallized aspect of silver nanoparticles. According to

Table 2 Effect of varying the concentration of NADPH on the PDI and HD of nano-Ag using 0.5 mM AgNO3 . (mM)

PDI

HD (nm)

0.5NADPH:0.5AgNO3 1 NADPH:0.5AgNO3 1.5 NADPH:0.5AgNO3

0.195 0.201 0.419

19.61 21.25 27.93

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Fig. 4. TEM images of nano-Ag synthesized by using variable concentrations of NADPH with 0.5 mM concentration of AgNO3 .

Sherrer’s equation, the Ag nanoparticles exhibit an average size of 20 nm, which is in good agreement with TEM and hydrodynamic diameters. It should be noted that this measurement is solely based on the (1 1 1) reflection, because of the weak intensity from other reflections.

3.2. Stability of nano-Ag colloidal solution All sets of batch experiments in the studied range of concentrations were stable aqueous dispersions and the formation of any solid precipitate was not observed even after 2 months storage at

C

analyse edx

1.0 Ag

cps/eV

0.8

0.6

0.4

Na

0.2

O P

S

0.0 1

2

3

4

5

6

KeV Fig. 5. EDX spectrum of nano-Ag.

7

8

9

10

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235

2.5 1 day

60 day

Absorbance

2

1.5

1

0.5

0 300

400

500

600

Wavelength (nm) Fig. 6. X-ray diffractogram of nano-Ag (brackets refer to Miller indices). Fig. 8. Absorption spectra of nano-Ag before and after storage at room temperature.

ambient conditions. Zeta potential (ZP) values reveal details about the surface charge and stability of the synthesized silver nanoparticles which was also supported by absorbance peaks in SPR. The results of the zeta potential values for nano-Ag are −41 ± 5 mV, indicating the stability of the synthesized nanoparticles (Fig. 7). At different solution concentrations, AgNO3 with an equimolar ratio of NADPH shows no significant variation in the zeta potential value of nano-Ag. In general, a value of 25 mV (positive or negative) can be taken as the arbitrary value that separates low-charged surfaces from highly charged surfaces, and colloids with a zeta potential from 40 to 60 mV are believed to have good stability [36,39]. The negative charge of silver nanoparticles can be attributed to adsorption of anions such as phosphate and hydroxide on the surface of silver nanoparticles, and the resulting electrostatic double layer repulsion that stabilizes the nanoparticle dispersion. The stability of the colloidal solution of nano-Ag was evaluated during a 2 month period. The stability was traced at different aging periods by UV–vis spectroscopy for samples diluted appropriately with de-ionized water. As it can be seen from Fig. 8, the intensity of the plasmon absorption band displayed no decrease even after 60 days. Also, the maximum absorbance peak wavelength was nearly the same (410 nm) for the 60 days aged sample. 3.3. Kinetics of particle formation As shown in Fig. 1A and B, upon increasing the silver nitrate concentration the absorption value increased gradually within the studied concentration range and it is noted that with an increase in time the peak become sharper. NADPH plays the role of both

1400000

-41±5 mV

Total Counts

1200000

3.4. Regeneration of NADPH

1000000 800000 600000 400000 200000 0 -200

reducing and stabilizing agent opening the avenues to study kinetics of particle formation and growth without the influence of an external capping agent. The relatively weak reducibility of NADPH resulted in a slow rate of the reduction process, which was a key factor to realize the controllable synthesis of silver nanocrystals [33]. For a kinetic characterization of particle formation, the light absorbance at 410 nm was plotted against time at various AgNO3 concentrations (Fig. 1A). The concentration 0.31 mM of AgNO3 has been selected to describe the characteristic sigmoid curve because the reaction was completed within the limit of the absorbance detection range of the spectrophotometer. The conversion of Ag+ ions to metallic Ag is relatively complete. It can be concluded from the fact that equimolar or excess concentrations of the reducing agent (NADPH) were supplied. The reaction was monitored till the wavelength of the surface plasmon resonance peak showed no further change. However, the direct measure of the AgNO3 concentrations will be targeted in our follow-up work. The kinetic curve started with a slow induction phase, then rose sharply and finally reach a constant saturation value, indicative of an autocatalytic reaction [40]. Bönnemann and Richards Ryan [41] described that the nucleation of metal nanoparticles prepared by reduction may be an autocatalytic process. Patakfalvi et al. [42] measured the kinetics of nano-Ag synthesis in a polymers stabilized suspension using a UV–vis spectrophotometer and transmission electron microscopy. They differentiated between the two main phases of particle formation, i.e. nucleation and growth and characterized their rates with the help of appropriate kinetic equations. Although it remains an interesting assumption for future exploration, we suspect that the first, slower phase corresponded to the nucleation, which did not cause a significant increase in absorbance, then followed by nucleus growth (Fig. 1A).

-100

0

100

200

Zeta Potenal Fig. 7. Zeta potential of nano-Ag prepared from the reaction mixture containing NADPH and AgNO3 in a 1:1 molar ratio.

Glucose-6-phosphate dehydrogenase has been insolubilized by covalent bonding to alumina membrane [34]. The amount of NADPH formed is calculated from the molar extinction coefficient. The molar extinction coefficient for NADPH at 340 nm is 6220 M−1 cm−1 [43]. The synthesized nanoparticles were removed by centrifugation. The supernatant containing NADP+ was supplemented with appropriate amount of glucose 6-phosphate and cross circulated through the alumina membrane containing immobilized G6PD at the flow rate of 50 ␮L/min leading to a 33% regeneration of NADPH. The rest of the NADPH might have remained absorbed by nano-Ag or was not available for regeneration for uncertain reasons. The regenerated NADPH was utilized

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again for the synthesis of the nano-Ag by adding an appropriate amount of AgNO3 .

NADP+ G6P

NADPH + H+ Gluconolactone-6-P

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