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Synthesis of gold and silver nanoparticles using purified URAK
Colloids and Surfaces B: Biointerfaces 86 (2011) 353–358
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Synthesis of gold and silver nanoparticles using purified URAK Venkataraman Deepak , Paneer Selvam Umamaheshwaran , Kandasamy Guhan , Raja Amrisa Nanthini , Bhaskar Krithiga, Nagoor Meeran Hasika Jaithoon, Sangiliyandi Gurunathan ∗ Division of Molecular and Cellular Biology, Department of Biotechnology, Kalasalingam University, Anand Nagar, Krishnankoil 626190, Tamil Nadu, India
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Article history: Received 4 March 2011 Received in revised form 8 April 2011 Accepted 8 April 2011 Available online 16 April 2011 Keywords: URAK AgNPs AuNPs AFM TEM
a b s t r a c t This study aims at developing a new eco-friendly process for the synthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) using purified URAK. URAK is a fibrinolytic enzyme produced by Bacillus cereus NK1. The enzyme was purified and used for the synthesis of AuNPs and AgNPs. The enzyme produced AgNPs when incubated with 1 mM AgNO3 for 24 h and AuNPs when incubated with 1 mM HAuCl4 for 60 h. But when NaOH was added, the synthesis was rapid and occurred within 5 min for AgNPs and 12 h for AuNPs. The synthesized nanoparticles were characterized by a peak at 440 nm and 550 nm in the UV–visible spectrum. TEM analysis showed that AgNPs of the size 60 nm and AuNPs of size 20 nm were synthesized. XRD confirmed the crystalline nature of the nanoparticles and AFM showed the morphology of the nanoparticle to be spherical. FT-IR showed that protein was responsible for the synthesis of the nanoparticles. This process is highly simple, versatile and produces AgNPs and AuNPs in environmental friendly manner. Moreover, the synthesized nanoparticles were found to contain immobilized enzyme. Also, URAK was tested on RAW 264.7 macrophage cell line and was found to be non-cytotoxic until 100 g/ml. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The recent advancements in the synthesis of nanoparticles and their applications in biology have led to the rise of “Nanobiotechnology” [1]. Recent reports depict that silver nanoparticles and gold nanoparticles have multitude applications. AgNPs were found to possess anti-microbial activity [2], anti-angiogenic activity [3], anti-permeability activity [4] and anti-cancerous activity [5]. Similarly AuNPs can be used in sensors, photoelectrochemical materials [6] and photocatalysts [7]. Medically, AuNPs have broader applications in gene delivery [8] and labeling [9]. Gold nanoparticles as nano shells have been used to treat cancer, for cancer detection [10]. Besides they can also be used as chromophores [11]. Although chemical methods are available for the synthesis of nanoparticles, they involve toxic chemicals, and need a stabilizer for preventing the agglomeration of the nanoparticles [12,13]. Chemically synthesized nanoparticles may contain the chemicals adsorbed on their surface which may also limit the use of nanoparticles in medical applications [13]. Moreover, the reducing agents used for the synthesis are not easily disposed off from the environment [14]. In the present scenario, numerous microorganisms have been reported to synthesize various nanoparticles. Previously, AgNPs
∗ Corresponding author. E-mail address: [email protected] (S. Gurunathan). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.04.019
have been shown to be synthesized by bacteria like Brevibacterium casei [15], E. coli [16], fungi like Fusarium oxysporum [17] and AuNPs by B. casei [15], F. oxysporum [18] etc., Biosynthesis of nanoparticles gained significance because of their relative ease in the synthesis. It does not require environmentally toxic chemicals for the synthesis of nanoparticles and size control can be easily obtained by controlling the pH and temperature of the environment [16]. Moreover, the proteins act as stabilizers and reduce the additional step of stabilizing the synthesized nanoparticles. But synthesis of nanoparticles by the biomass also contains some drawbacks. The major problem encountered is the isolation and purification of the nanoparticle from the biomass. This requires large number of downstream processing steps including sonication, ultracentrifugation to arrive at the maximum yield [15,16]. Moreover, there is a chance for the presence of endotoxin in the nanoparticles which may limit the use of nanoparticles in the medical field [19]. Nearly last two decades have witnessed the use of microbial fibrinolytic enzymes in augmenting thrombosis therapy. But, still searches have been made to identify various potential fibrinolytic enzymes from various sources with higher efficiency. In this work we tried to synthesize AuNPs and AgNPs using the enzyme URAK, isolated and purified from the supernatant of the organism B. cereus NK1. URAK is essentially isolated as a fibrinolytic enzyme and found to be stable in presence of Fe3+ [20]. Previously, the enzyme nitrate reductase has been used to synthesize silver nanoparticles from silver nitrate. Several aids have been supplemented at this juncture in synthesizing AgNPs like NADH, and a stabilizer [21] and
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␣-amylase has been used to synthesize AuNPs [22]. But here, we directly synthesized AgNPs by incubating silver nitrate with purified URAK and AuNPs by incubating with HAuCl4 . Nanoparticles synthesized using URAK were characterized using UV–visible spectroscopy, XRD analysis, transmission electron microscopy, FT-IR and atomic force microscopy. The nanoparticles synthesized were found to contain the enzyme immobilized on it when incubated in NaOH free medium. And the immobilized URAK were found to possess enhanced thermostability and pH stability. 2. Materials and methods 2.1. Organism and culture conditions URAK was essentially produced by B. cereus NK1 (GU167978). Working cultures were prepared by streaking a colony of the organism in nutrient agar plates and sub-cultured every fortnight and pure colonies were isolated and stored at −80 ◦ C. 2.2. Production and purification of URAK URAK was produced in the optimized medium that has been reported previously. It comprises of 0.5% glucose, 0.5% soybean meal, 0.5% CaCl2 and 0.2% MgSO4 . Produced enzyme was purified by acetone precipitation and cation exchange Chromatography [20]. 2.3. Purification of URAK After 24 h of incubation, the supernatant was separated by centrifugation (6000×g, 10 min) and precipitated with ice-cold acetone. The obtained precipitate was resuspended over a 50 mM Tris-HCl buffer pH 6.5. The concentrated supernatant was added to a CM-methacrylite column (Bio-Rad, USA), which was preequilibrated with 50 mM Tris-HCl buffer at pH 6.5. The bound URAK was eluted with 0.2 M NaCl. Active fractions were pooled and used as the purified enzyme solution. The amount of protein was estimated based on the Lowry’s method and the homogeneity was tested with SDS-PAGE analysis was performed based on the method of Laemmli. 2.4. Synthesis of AgNPs and AuNPs using URAK AgNPs has been synthesized by incubating 1 mg of purified URAK in 10 ml of Tris-HCl buffer pH 9 containing 1 mM AgNO3 at 37 ◦ C. The reaction mixture was divided into two. One containing NaOH and another is NaOH free. Similarly AuNPs are synthesized by incubating 1 mg of purified URAK in 10 ml of Tris-HCl buffer pH 9 containing 1 mM HAuCl4. Similar to AgNP synthesis the reaction mixture was divided into two, one with NaOH and another without NaOH.
2.5.3. XRD analysis In order to determine the nature of particles, AgNPs and AuNPs synthesized using URAK were centrifuged and washed and air dried. The air dried powder was then subjected to XRD analysis on a PANalytical XRD analyzer (X’pert PRO) operating in transmission mode at 40 kV and 30 mA with Cu K radiation. 2.5.4. Atomic force microscopy A thin film of the sample was prepared on a glass slide by dropping 100 l of the sample on the slide, and was allowed to dry for 5 min. The slides were then scanned with the AFM. The experiments are conducted with a The Nanosurf easyScan 2 FlexAFM Atomic Force Microscope. The AFM characterization was carried out at ambient temperature in non-contact mode using silicon nitride tips with varying resonance frequencies at a linear scanning rate of 0.5 Hz. 2.5.5. Fourier transform infrared (FTIR) spectroscopy analysis The dried sample was also subjected to FTIR Spectroscopy analysis. Two milligrams of the sample was mixed with 200 mg KBr (FT-IR grade) and pressed into a pellet. The sample pellet was placed into the sample holder and FT-IR spectra were recorded in FT-IR spectroscopy at a resolution of 4 cm−1 . 2.5.6. Effect of pH and temperature on enzyme stability Analysis of the effect of pH level on enzyme stability was carried out by incubating the free and immobilized enzyme for 1 h at 37 ◦ C in the following buffers: Acetate, phosphate, Tris-HCl, sodium carbonate and glycine–NaOH buffers. All buffers were 0.1 M. For measuring its thermal stability, the free and immobilized enzyme was incubated in 25 mM sodium phosphate buffer at pH 7.0 for 30 min at 30–80 ◦ C. After incubation, the residual activity was determined. 2.5.7. Cell viability assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay was used to determine the viability of the cells according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). The assay is based on the reduction of the MTT by the mitochondrial dehydrogenase resulting in a blue formazan product that can be measured in a multiwell spectrophotometer (Biorad, model 680, Japan). Briefly, 2 × 103 cells of macrophages (RAW 264.7) were seeded in 96-well microtiter plates and starved for 6 h in serum free medium. To determine the effect of URAK on cell viability of seeded macrophages, serum starved cells were treated with different concentrations of URAK (10 ng/ml, 100 ng/ml, 1 g/ml, 10 g/ml, 100 g/ml) incubated for 24 h in 5% CO2 at 37 ◦ C. After incubation 10 l of MTT was added to each well and further incubated for 4 h. 100 l of dissolving buffer was used to dissolve the formed crystals and the absorbance was read at 595 nm. All the experiments were repeated thrice for consistency.
2.5. Characterization of nanoparticles
3. Results
2.5.1. UV–visible spectroscopic analysis 1 ml of sample supernatant was withdrawn and absorbance was measured by using UV–visible spectrophotometer. UV–visible analysis was performed in a Shimadzu-UV–visible spectrophotometer, (model 9200) with a resolution of 0.72 nm.
3.1. Synthesis of AgNPs and AuNPs using URAK and its characterization
2.5.2. Transmission electron microscopy (TEM) measurements Sample for TEM analysis was prepared by loading a small volume of AgNPs and AuNPs on carbon-coated copper grids and solvent was allowed to evaporate for 30 min. TEM measurements were performed on JOEL model instrument 1200 EX instrument on carbon coated copper grids with an accelerating voltage of 80 kV.
Incubation of 1 mM AgNO3 with URAK synthesized AgNPs. The culture did not result in the synthesis of nanoparticle when the pH was acidic. But when the pH of the solution is in the alkaline region, URAK synthesized nanoparticles within 24 h of incubation. This showed that URAK has an inherent capacity to synthesize AgNPs from AgNO3 . Interestingly, when 1 ml of 1 M solution of NaOH was added to the reaction mixture the colour change was immediate and AgNPs were synthesized with in a period of 5 min. Fig. 1 shows the change of colour with in 5 min after the addition of NaOH to
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Fig. 1. Visual observation of nanoparticle synthesis. The nanoparticles were synthesized using URAK. In figure, (F) is the control, (A) is the tube containing AuNPs synthesized using URAK and (B) is the tube containing AgNPs synthesized using URAK. All the reactions were carried out in Tris HCl buffer pH 9.0.
the reaction mixture. The formation of brown colour can be designated to the formation of the AgNPs in the reaction mixture. The colour formation in the reaction mixture is due to the excitations of the surface plasmon resonance of the AgNPs formed in the reaction mixture. Previous reports have shown that addition of NaOH enhances the reaction rate during the production of silver nanoparticles. Similarly, Incubation of 1 mM HAuCl4 with URAK resulted in the synthesis of AuNPs. But unlike AgNPs, the synthesis of AuNPs took a longer time of 60 h. URAK besides synthesizing AgNPs, is also capable of synthesis of AuNPs. This reaction was also speeded up by the addition of NaOH. When NaOH was added to the reaction mixture the synthesis was quick and occurred with in 20 h of incubation. The synthesis of AuNPs was primarily confirmed by the formation of purple colour in the reaction mixture (Fig. 1) due to the surface plasmon resonance.
3.2. UV–visible spectroscopic analysis After the primary characterization with colour change the reaction mixture was subjected to UV–visible spectroscopy measurement. UV–visible spectroscopy is one of the imperative techniques which can ascertain the synthesis of metal nanoparticles. The synthesized AgNPs is shown to have peak intensity at 440 nm and AuNPs at 550 nm. Fig. 2A shows the UV–visible spectrum of AgNPs synthesized without the addition of NaOH. Fig. 2B shows the spectrum of AuNPs synthesized without the addition of NaOH to the reaction mixture. For AgNPs, the NaOH free (NF) reaction mixture the intensity at 440 nm steadily increased and reached maximum after 24 h (Fig. 2A). Whereas the in the NaOH containing (NC) mixture the peak obtained was immediate and the intensity of the peak is comparable with the NF mixture at 24 h (data not shown). Similarly, the peak value at 550 nm slowly increased up to 60 h (Fig. 2B) whereas the peak obtained at 20th hour in the NaOH containing mixture was comparable with the NF mixture (data not shown).
Fig. 2. UV–visible analysis of AgNP synthesis. (A) shows the AgNPs synthesis in NF medium which shows a peak at 440 nm. The peak intensity increased with time and it reached maximum at 24 h. (B) shows the AuNPs synthesized using NF medium where the peak obtained was at 450 nm and the peak intensity increased with time up to 60 h.
3.3. TEM analysis TEM is considered to be an important tool to analyze the formation of metal nanoparticles. A representative of TEM recorded from the AgNPs and AuNPs film deposited on a carbon coated copper grid is shown in Fig 3. Fig. 3A contains the picture of AgNPs synthesized by reduction of Ag+ ions by URAK and Fig. 3B contains the picture of AuNPs synthesized by the reduction of Au3+ ions by URAK. The size of AgNPs synthesized was relatively larger is in the range of 50–80 nm and most of the synthesized AgNPs has the size of 60 nm. The nanoparticle was found to be highly dispersed and of spherical in morphology. AuNPs were synthesized in the range of 15–30 nm with an average of 20 nm. AuNPs also had a spherical morphology and found to be highly dispersed. 3.4. X-ray diffraction studies Fig. 4A shows the XRD pattern obtained for AgNPs synthesized by URAK in NF mixture. There are three Bragg reflections at 37.87, 45.40, 64.59 which corresponds to the (1 1 1), (2 0 0) and (2 2 0) planes which can be indexed to the FCC AgNPs based on the comparison with the standard as given by JCPDS (file nos. 04-0783 and 84-0713). Therefore based on the XRD patterns it can be confirmed that the synthesized AgNPs from URAK are of crystalline nature. Debye-Scherrer’s equation was used to calculate the mean size of AgNPs using the FWHM values at the (1 1 1) plane which showed that the size of the AgNPs synthesized is in the range of 60 nm. Fig. 4B shows the XRD pattern of AuNPs synthesized using URAK in NC mixture. There is only one brag reflection at 38.10◦ that corresponds to the (1 1 1) plane which can be indexed to FCC AuNPs when compared with the standard given by JCPDS (file no. 01-1174 for Au). Based on the Debye-Scherrer’s equation the size of the AuNPs
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Fig. 4. XRD spectra of AgNPs (A) and AuNPs (B) synthesized by URAK at NF condition with pH 9. Fig. 3. TEM image of AgNPs and AuNPs synthesized using URAK. (A) shows the representative TEM image of AgNPs synthesized using URAK and (B) shows the representative TEM image of AuNPs. The size of the particle was in the range 60 nm for AgNPs and 20 nm for AuNPs.
was found to be 20 nm which shows that AuNPs are of crystalline in nature. 3.5. Analysis of AgNPs in AFM To determine the topography of the AgNPs synthesized using URAK, the sample was dried and the surface was scanned in
an area of 2.36 m × 2.36 m. Typical AFM image of both AgNPs and AuNPs were obtained and are presented in Fig. 5. Fig. 5A shows the AFM image of AgNPs and Fig. 5B shows the AFM image of AuNPs. In Fig. 5A, the image demonstrates the spherical nature of the AgNP single crystal and the size obtained was also similar to 50–60 nm. Similarly Fig. 5B shows the AuNPs synthesized using URAK. The dried sample was scanned in an area of 2.94 m × 2.94 m. Although gold nanoparticles are clustered, the spherical morphology of the AuNPs can be easily observed.
Fig. 5. AFM analysis. (A) shows the AFM image of AgNPs synthesized using URAK where a single particle can be seen with the spherical morphology and (B) shows the AFM image of the AuNPs synthesized using URAK where also the spherical nature can be observed.
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Fig. 6. Effect of URAK on cell viability. MTT assay was used to check the effect of URAK on the viability of macrophage. Among all the concentration tested URAK did not significantly induce any cytotoxicity in cells. Data presented is the mean of three different experiments.
3.6. FT-IR analysis The synthesized nanoparticle was dried and subjected to FT-IR studies in order to determine the type of responsible biomolecules that aided the synthesized AgNPs. Fig. 6 shows the FTIR spectra of AgNPs synthesized using purified URAK. Two peaks at 1039 and 1458 which are the stretches due to C–O–C & C–OH and C C stretching respectively which corroborates with earlier reports [26,27]. Therefore, it can be confirmed that proteins are responsible for reduction of Ag+ ions. In the case of AuNPs synthesis FT-IR analysis showed two peaks at 1025, 1220, 1622, 1738 which corresponds to the C–N stretching in aliphatic amines, amide III and Amide I bands of proteins and carbonyl groups respectively [28] (data not shown). 3.7. Cytotoxic analysis of URAK In order to determine the effect of different concentration of URAK on the viability of cells, macrophage (RAW 264.7) was used. In all the concentration tested, the viability of macrophages, URAK did not decrease viability in any of the concentration tested (10 ng/ml–100 g/ml) significantly. 4. Discussion This manuscript describes the use of URAK in synthesizing AgNPs and AuNPs. URAK is a fibrinolytic enzyme isolated and purified from the culture supernatant of B. cereus NK1. The enzyme was produced in the optimized medium and purified as reported. The fractions that contained the fibrinolytic activity and homogeneity in SDS-PAGE were pooled and used for further experiments [20]. To the best of our knowledge this is the first report claiming the synthesis of both the nanoparticles by a single enzyme. Previously biosynthesis of nanoparticles has been reported by various organisms and plants including F. oxysporum [17], B. casei [16], Magnolia leaf broth [32] etc. This method was previously considered to be advantageous as they prevented the use of various toxic chemical reducing agents like sodium borohydride (NaBH4 ) and hydrazine (NH2 –NH2 ) These extremely reactive chemicals does not only possess environmental but also biological risks too [33]. But, microbial synthesis also has the possibility of presence of toxic substances like endotoxin on the surface of the nanoparticles. The contaminants present with the nanoparticle may hinder the use of nanoparticles in various biological applications, which requires the synthesized nanoparticle to be highly purified from the immunomodulating contaminants present [19]. Synthesis of NPs from culture supernatants may be little more advantageous
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than using the biomass, but the problem arises during the purification of the particles from the other molecules present on the culture supernatant [17]. Since macrophages are one of the immune cells first encounter the foreign materials the effect of URAK was checked in it and the enzyme did not induce toxicity up to the concentration of 100 g/ml. Therefore synthesis of nanoparticles by specific enzyme which is non toxic to the mammalian cells is highly advantageous and this would greatly reduce the downstream processing steps in obtaining pure nanoparticles for various applications. Moreover, the physical and chemical methods for the production at largescale usually results in larger particles whereas the biosynthesis can be effectively used for the production of smaller nanoparticles at large-scales operations [31]. Purification of nanoparticles from the contaminations remains a great problem during biosynthesis. Hence, we report here the synthesis of nanoparticles using eco-friendly methods, which may prevent the contamination of the environment by both the toxic chemicals and other biological substances. Here, purified URAK also act as a capping agent too which also prevents the agglomeration of the synthesized AgNPs and AuNPs. In chemical synthesis this process is an additional step. When bacteria or other biomass are used for the synthesis of nanoparticles, most of the particle adhere to the biomass itself and great efforts have to be put forth for the down stream processing steps [15]. If not during disposal into the environment the synthesized nanoparticles may accumulate in the environment affecting the microbial diversity, as AgNPs were shown to have potential anti-microbial activity and AuNPs are relatively costlier. Since we have used purified URAK without the addition of any other agents it can be concluded that URAK has the ability to synthesize AgNPs and AuNPs. Previously nitrate reductase mediated AgNPs synthesis resulted in the nanoparticle size range of 10–25 nm and a capping peptide is added to stabilize the synthesized nanoparticles [21]. Besides nitrate reductase, Bovine serum albumin and various extracellular fractions have been reported to synthesize AgNPs [17,29]. Some of the previous reports have shown that increasing the alkalinity of the reaction mixture, the formation of AgNPs is rapid [16]. During our experiment also increasing the alkalinity resulted in faster synthesis of nanoparticles. Similarly, AuNPs have been previously synthesized using the enzyme ␣-amylase and the activity of ␣-amylase was not affected after the synthesis of nanoparticles [22]. Protease enzyme from Actinobacter sp. has been reported to aid the synthesis of AuNPs [30]. However, BSA did not aid the synthesis of AuNPs when incubated with HAuCl4 alone [29]. Here the pure enzyme URAK aided the synthesis of both AuNPs and AgNPs which was also found to bind with the nanoparticle synthesized, becoming a stabilizer. Moreover, when nanoparticles synthesized using NaOH free mixture was analyzed, it retained the activity of the enzyme. Primarily the synthesis was characterized by UV–visible spectroscopic measurements. This corresponds with the previous reports where AgNPs and AuNPs were shown to have peak intensities in the range of 440 nm [16,23] and 540 nm [24] respectively. Moreover, increasing the pH enhanced the rate of synthesis of nanoparticle synthesis. This greatly corroborates with the previous conditions that alkaline conditions favors the synthesis of silver nanoparticles [10,25]. Presence of OH− ions reduced the time for AgNPs and AuNPs synthesized, whereas when the concentration of hydroxide ion was lesser, the time taken for the synthesis of AgNPs and AuNPs was very much longer. This shows that addition of OH− ions factious the nucleation resulting in the rapid synthesis of nanoparticles [16]. Although nanoparticles are bound to the enzyme, the presence of amino acids, especially tryptophan and tyrosine are previously reported to possess capacity to reduce various metal ions [29]. A
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careful genome analysis showed that B. cereus contains a gene for alkaline serine protease, that codes for protein with a length of 397 amino acids (GI: 225785743). The approximate molecular weight calculated from the sequence is found to be 43 kDa, which is very close to the obtained molecular weight of the purified URAK from B. cereus NK1. The amino acid sequence present in the protein contains 5 tryptophan residues and 23 tyrosine residues. These amino acids not only reduce the metal ions but also reported to cap the metal nanoparticles formed. Besides synthesis of nanoparticles, in NF mixture, both the particles contained enzymes with very good activity. Both the AuNPs and AgNPs synthesized had been checked for fibrinolytic activity and it was present. This was compared with biologically synthesized nanoparticles which did not exhibit any activity. Therefore, besides synthesis of AuNPs and AgNPs, the protein was found to bind on them and both the nanoparticles were checked for thermo and pH stability. Both the nanoparticles enhanced the stability of the immobilized protein. Therefore this might be an easy method for immobilization of fibrinolytic URAK. Previous methods have used a linker to immobilize the protein where the nanoparticles were synthesized separately, using chemical methods and linked with the protein. Here the nanoparticles were synthesized and URAK was immobilized with relative ease. Previously, various enzymes have been immobilized on both AuNPs and AgNPs. One such enzyme is Glucose oxidase. Glucose oxidase has been immobilized both on AuNPs and AgNPs with different linkers like carbodiimide linker and 6-aminohexanoic acid for the purpose of biosensor fabrication. Both the methods have been found to enhance the stability of the enzyme [34,35]. For immobilization of glucose oxidase, the nanoparticles were synthesized separately, purified enzyme separately and linked by linkers. But with URAK, no linkers have been used and the enzyme itself synthesized nanoparticles. 5. Conclusion The reduction of Ag+ and Au3+ ions by purified URAK has been reported here. The reduction resulted in the formation of AgNPs and AuNPs of spherical morphology. Here URAK not only synthesized nanoparticles but it remained bound with the nanoparticles as a capping agent thus preventing the agglomeration of the nanoparticles. This method may be highly useful as this method can be easily scaled-up and the eco-friendly nature of the nanoparticle synthesis methodology is one of the advantages. Moreover, the yield obtained can be the maximum as pure nanoparticles are present without any contaminants. Acknowledgements The author V. Deepak gratefully acknowledges Council for Scientific and Industrial Research (CSIR), India for providing Senior Research Fellowship (reference number: 9/1012(0001)/2k10-EMRI). We also thank Prof. Pushpa Viswanathan, Cancer Institute (WIA),
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Synthesis of gold and silver nanoparticles using purified URAK Venkataraman Deepak , Paneer Selvam Umamaheshwaran , Kandasamy Guhan , Raja Amrisa Nanthini , Bhaskar Krithiga, Nagoor Meeran Hasika Jaithoon, Sangiliyandi Gurunathan ∗ Division of Molecular and Cellular Biology, Department of Biotechnology, Kalasalingam University, Anand Nagar, Krishnankoil 626190, Tamil Nadu, India
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Article history: Received 4 March 2011 Received in revised form 8 April 2011 Accepted 8 April 2011 Available online 16 April 2011 Keywords: URAK AgNPs AuNPs AFM TEM
a b s t r a c t This study aims at developing a new eco-friendly process for the synthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) using purified URAK. URAK is a fibrinolytic enzyme produced by Bacillus cereus NK1. The enzyme was purified and used for the synthesis of AuNPs and AgNPs. The enzyme produced AgNPs when incubated with 1 mM AgNO3 for 24 h and AuNPs when incubated with 1 mM HAuCl4 for 60 h. But when NaOH was added, the synthesis was rapid and occurred within 5 min for AgNPs and 12 h for AuNPs. The synthesized nanoparticles were characterized by a peak at 440 nm and 550 nm in the UV–visible spectrum. TEM analysis showed that AgNPs of the size 60 nm and AuNPs of size 20 nm were synthesized. XRD confirmed the crystalline nature of the nanoparticles and AFM showed the morphology of the nanoparticle to be spherical. FT-IR showed that protein was responsible for the synthesis of the nanoparticles. This process is highly simple, versatile and produces AgNPs and AuNPs in environmental friendly manner. Moreover, the synthesized nanoparticles were found to contain immobilized enzyme. Also, URAK was tested on RAW 264.7 macrophage cell line and was found to be non-cytotoxic until 100 g/ml. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The recent advancements in the synthesis of nanoparticles and their applications in biology have led to the rise of “Nanobiotechnology” [1]. Recent reports depict that silver nanoparticles and gold nanoparticles have multitude applications. AgNPs were found to possess anti-microbial activity [2], anti-angiogenic activity [3], anti-permeability activity [4] and anti-cancerous activity [5]. Similarly AuNPs can be used in sensors, photoelectrochemical materials [6] and photocatalysts [7]. Medically, AuNPs have broader applications in gene delivery [8] and labeling [9]. Gold nanoparticles as nano shells have been used to treat cancer, for cancer detection [10]. Besides they can also be used as chromophores [11]. Although chemical methods are available for the synthesis of nanoparticles, they involve toxic chemicals, and need a stabilizer for preventing the agglomeration of the nanoparticles [12,13]. Chemically synthesized nanoparticles may contain the chemicals adsorbed on their surface which may also limit the use of nanoparticles in medical applications [13]. Moreover, the reducing agents used for the synthesis are not easily disposed off from the environment [14]. In the present scenario, numerous microorganisms have been reported to synthesize various nanoparticles. Previously, AgNPs
∗ Corresponding author. E-mail address: [email protected] (S. Gurunathan). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.04.019
have been shown to be synthesized by bacteria like Brevibacterium casei [15], E. coli [16], fungi like Fusarium oxysporum [17] and AuNPs by B. casei [15], F. oxysporum [18] etc., Biosynthesis of nanoparticles gained significance because of their relative ease in the synthesis. It does not require environmentally toxic chemicals for the synthesis of nanoparticles and size control can be easily obtained by controlling the pH and temperature of the environment [16]. Moreover, the proteins act as stabilizers and reduce the additional step of stabilizing the synthesized nanoparticles. But synthesis of nanoparticles by the biomass also contains some drawbacks. The major problem encountered is the isolation and purification of the nanoparticle from the biomass. This requires large number of downstream processing steps including sonication, ultracentrifugation to arrive at the maximum yield [15,16]. Moreover, there is a chance for the presence of endotoxin in the nanoparticles which may limit the use of nanoparticles in the medical field [19]. Nearly last two decades have witnessed the use of microbial fibrinolytic enzymes in augmenting thrombosis therapy. But, still searches have been made to identify various potential fibrinolytic enzymes from various sources with higher efficiency. In this work we tried to synthesize AuNPs and AgNPs using the enzyme URAK, isolated and purified from the supernatant of the organism B. cereus NK1. URAK is essentially isolated as a fibrinolytic enzyme and found to be stable in presence of Fe3+ [20]. Previously, the enzyme nitrate reductase has been used to synthesize silver nanoparticles from silver nitrate. Several aids have been supplemented at this juncture in synthesizing AgNPs like NADH, and a stabilizer [21] and
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␣-amylase has been used to synthesize AuNPs [22]. But here, we directly synthesized AgNPs by incubating silver nitrate with purified URAK and AuNPs by incubating with HAuCl4 . Nanoparticles synthesized using URAK were characterized using UV–visible spectroscopy, XRD analysis, transmission electron microscopy, FT-IR and atomic force microscopy. The nanoparticles synthesized were found to contain the enzyme immobilized on it when incubated in NaOH free medium. And the immobilized URAK were found to possess enhanced thermostability and pH stability. 2. Materials and methods 2.1. Organism and culture conditions URAK was essentially produced by B. cereus NK1 (GU167978). Working cultures were prepared by streaking a colony of the organism in nutrient agar plates and sub-cultured every fortnight and pure colonies were isolated and stored at −80 ◦ C. 2.2. Production and purification of URAK URAK was produced in the optimized medium that has been reported previously. It comprises of 0.5% glucose, 0.5% soybean meal, 0.5% CaCl2 and 0.2% MgSO4 . Produced enzyme was purified by acetone precipitation and cation exchange Chromatography [20]. 2.3. Purification of URAK After 24 h of incubation, the supernatant was separated by centrifugation (6000×g, 10 min) and precipitated with ice-cold acetone. The obtained precipitate was resuspended over a 50 mM Tris-HCl buffer pH 6.5. The concentrated supernatant was added to a CM-methacrylite column (Bio-Rad, USA), which was preequilibrated with 50 mM Tris-HCl buffer at pH 6.5. The bound URAK was eluted with 0.2 M NaCl. Active fractions were pooled and used as the purified enzyme solution. The amount of protein was estimated based on the Lowry’s method and the homogeneity was tested with SDS-PAGE analysis was performed based on the method of Laemmli. 2.4. Synthesis of AgNPs and AuNPs using URAK AgNPs has been synthesized by incubating 1 mg of purified URAK in 10 ml of Tris-HCl buffer pH 9 containing 1 mM AgNO3 at 37 ◦ C. The reaction mixture was divided into two. One containing NaOH and another is NaOH free. Similarly AuNPs are synthesized by incubating 1 mg of purified URAK in 10 ml of Tris-HCl buffer pH 9 containing 1 mM HAuCl4. Similar to AgNP synthesis the reaction mixture was divided into two, one with NaOH and another without NaOH.
2.5.3. XRD analysis In order to determine the nature of particles, AgNPs and AuNPs synthesized using URAK were centrifuged and washed and air dried. The air dried powder was then subjected to XRD analysis on a PANalytical XRD analyzer (X’pert PRO) operating in transmission mode at 40 kV and 30 mA with Cu K radiation. 2.5.4. Atomic force microscopy A thin film of the sample was prepared on a glass slide by dropping 100 l of the sample on the slide, and was allowed to dry for 5 min. The slides were then scanned with the AFM. The experiments are conducted with a The Nanosurf easyScan 2 FlexAFM Atomic Force Microscope. The AFM characterization was carried out at ambient temperature in non-contact mode using silicon nitride tips with varying resonance frequencies at a linear scanning rate of 0.5 Hz. 2.5.5. Fourier transform infrared (FTIR) spectroscopy analysis The dried sample was also subjected to FTIR Spectroscopy analysis. Two milligrams of the sample was mixed with 200 mg KBr (FT-IR grade) and pressed into a pellet. The sample pellet was placed into the sample holder and FT-IR spectra were recorded in FT-IR spectroscopy at a resolution of 4 cm−1 . 2.5.6. Effect of pH and temperature on enzyme stability Analysis of the effect of pH level on enzyme stability was carried out by incubating the free and immobilized enzyme for 1 h at 37 ◦ C in the following buffers: Acetate, phosphate, Tris-HCl, sodium carbonate and glycine–NaOH buffers. All buffers were 0.1 M. For measuring its thermal stability, the free and immobilized enzyme was incubated in 25 mM sodium phosphate buffer at pH 7.0 for 30 min at 30–80 ◦ C. After incubation, the residual activity was determined. 2.5.7. Cell viability assay The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay was used to determine the viability of the cells according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). The assay is based on the reduction of the MTT by the mitochondrial dehydrogenase resulting in a blue formazan product that can be measured in a multiwell spectrophotometer (Biorad, model 680, Japan). Briefly, 2 × 103 cells of macrophages (RAW 264.7) were seeded in 96-well microtiter plates and starved for 6 h in serum free medium. To determine the effect of URAK on cell viability of seeded macrophages, serum starved cells were treated with different concentrations of URAK (10 ng/ml, 100 ng/ml, 1 g/ml, 10 g/ml, 100 g/ml) incubated for 24 h in 5% CO2 at 37 ◦ C. After incubation 10 l of MTT was added to each well and further incubated for 4 h. 100 l of dissolving buffer was used to dissolve the formed crystals and the absorbance was read at 595 nm. All the experiments were repeated thrice for consistency.
2.5. Characterization of nanoparticles
3. Results
2.5.1. UV–visible spectroscopic analysis 1 ml of sample supernatant was withdrawn and absorbance was measured by using UV–visible spectrophotometer. UV–visible analysis was performed in a Shimadzu-UV–visible spectrophotometer, (model 9200) with a resolution of 0.72 nm.
3.1. Synthesis of AgNPs and AuNPs using URAK and its characterization
2.5.2. Transmission electron microscopy (TEM) measurements Sample for TEM analysis was prepared by loading a small volume of AgNPs and AuNPs on carbon-coated copper grids and solvent was allowed to evaporate for 30 min. TEM measurements were performed on JOEL model instrument 1200 EX instrument on carbon coated copper grids with an accelerating voltage of 80 kV.
Incubation of 1 mM AgNO3 with URAK synthesized AgNPs. The culture did not result in the synthesis of nanoparticle when the pH was acidic. But when the pH of the solution is in the alkaline region, URAK synthesized nanoparticles within 24 h of incubation. This showed that URAK has an inherent capacity to synthesize AgNPs from AgNO3 . Interestingly, when 1 ml of 1 M solution of NaOH was added to the reaction mixture the colour change was immediate and AgNPs were synthesized with in a period of 5 min. Fig. 1 shows the change of colour with in 5 min after the addition of NaOH to
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Fig. 1. Visual observation of nanoparticle synthesis. The nanoparticles were synthesized using URAK. In figure, (F) is the control, (A) is the tube containing AuNPs synthesized using URAK and (B) is the tube containing AgNPs synthesized using URAK. All the reactions were carried out in Tris HCl buffer pH 9.0.
the reaction mixture. The formation of brown colour can be designated to the formation of the AgNPs in the reaction mixture. The colour formation in the reaction mixture is due to the excitations of the surface plasmon resonance of the AgNPs formed in the reaction mixture. Previous reports have shown that addition of NaOH enhances the reaction rate during the production of silver nanoparticles. Similarly, Incubation of 1 mM HAuCl4 with URAK resulted in the synthesis of AuNPs. But unlike AgNPs, the synthesis of AuNPs took a longer time of 60 h. URAK besides synthesizing AgNPs, is also capable of synthesis of AuNPs. This reaction was also speeded up by the addition of NaOH. When NaOH was added to the reaction mixture the synthesis was quick and occurred with in 20 h of incubation. The synthesis of AuNPs was primarily confirmed by the formation of purple colour in the reaction mixture (Fig. 1) due to the surface plasmon resonance.
3.2. UV–visible spectroscopic analysis After the primary characterization with colour change the reaction mixture was subjected to UV–visible spectroscopy measurement. UV–visible spectroscopy is one of the imperative techniques which can ascertain the synthesis of metal nanoparticles. The synthesized AgNPs is shown to have peak intensity at 440 nm and AuNPs at 550 nm. Fig. 2A shows the UV–visible spectrum of AgNPs synthesized without the addition of NaOH. Fig. 2B shows the spectrum of AuNPs synthesized without the addition of NaOH to the reaction mixture. For AgNPs, the NaOH free (NF) reaction mixture the intensity at 440 nm steadily increased and reached maximum after 24 h (Fig. 2A). Whereas the in the NaOH containing (NC) mixture the peak obtained was immediate and the intensity of the peak is comparable with the NF mixture at 24 h (data not shown). Similarly, the peak value at 550 nm slowly increased up to 60 h (Fig. 2B) whereas the peak obtained at 20th hour in the NaOH containing mixture was comparable with the NF mixture (data not shown).
Fig. 2. UV–visible analysis of AgNP synthesis. (A) shows the AgNPs synthesis in NF medium which shows a peak at 440 nm. The peak intensity increased with time and it reached maximum at 24 h. (B) shows the AuNPs synthesized using NF medium where the peak obtained was at 450 nm and the peak intensity increased with time up to 60 h.
3.3. TEM analysis TEM is considered to be an important tool to analyze the formation of metal nanoparticles. A representative of TEM recorded from the AgNPs and AuNPs film deposited on a carbon coated copper grid is shown in Fig 3. Fig. 3A contains the picture of AgNPs synthesized by reduction of Ag+ ions by URAK and Fig. 3B contains the picture of AuNPs synthesized by the reduction of Au3+ ions by URAK. The size of AgNPs synthesized was relatively larger is in the range of 50–80 nm and most of the synthesized AgNPs has the size of 60 nm. The nanoparticle was found to be highly dispersed and of spherical in morphology. AuNPs were synthesized in the range of 15–30 nm with an average of 20 nm. AuNPs also had a spherical morphology and found to be highly dispersed. 3.4. X-ray diffraction studies Fig. 4A shows the XRD pattern obtained for AgNPs synthesized by URAK in NF mixture. There are three Bragg reflections at 37.87, 45.40, 64.59 which corresponds to the (1 1 1), (2 0 0) and (2 2 0) planes which can be indexed to the FCC AgNPs based on the comparison with the standard as given by JCPDS (file nos. 04-0783 and 84-0713). Therefore based on the XRD patterns it can be confirmed that the synthesized AgNPs from URAK are of crystalline nature. Debye-Scherrer’s equation was used to calculate the mean size of AgNPs using the FWHM values at the (1 1 1) plane which showed that the size of the AgNPs synthesized is in the range of 60 nm. Fig. 4B shows the XRD pattern of AuNPs synthesized using URAK in NC mixture. There is only one brag reflection at 38.10◦ that corresponds to the (1 1 1) plane which can be indexed to FCC AuNPs when compared with the standard given by JCPDS (file no. 01-1174 for Au). Based on the Debye-Scherrer’s equation the size of the AuNPs
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Fig. 4. XRD spectra of AgNPs (A) and AuNPs (B) synthesized by URAK at NF condition with pH 9. Fig. 3. TEM image of AgNPs and AuNPs synthesized using URAK. (A) shows the representative TEM image of AgNPs synthesized using URAK and (B) shows the representative TEM image of AuNPs. The size of the particle was in the range 60 nm for AgNPs and 20 nm for AuNPs.
was found to be 20 nm which shows that AuNPs are of crystalline in nature. 3.5. Analysis of AgNPs in AFM To determine the topography of the AgNPs synthesized using URAK, the sample was dried and the surface was scanned in
an area of 2.36 m × 2.36 m. Typical AFM image of both AgNPs and AuNPs were obtained and are presented in Fig. 5. Fig. 5A shows the AFM image of AgNPs and Fig. 5B shows the AFM image of AuNPs. In Fig. 5A, the image demonstrates the spherical nature of the AgNP single crystal and the size obtained was also similar to 50–60 nm. Similarly Fig. 5B shows the AuNPs synthesized using URAK. The dried sample was scanned in an area of 2.94 m × 2.94 m. Although gold nanoparticles are clustered, the spherical morphology of the AuNPs can be easily observed.
Fig. 5. AFM analysis. (A) shows the AFM image of AgNPs synthesized using URAK where a single particle can be seen with the spherical morphology and (B) shows the AFM image of the AuNPs synthesized using URAK where also the spherical nature can be observed.
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Fig. 6. Effect of URAK on cell viability. MTT assay was used to check the effect of URAK on the viability of macrophage. Among all the concentration tested URAK did not significantly induce any cytotoxicity in cells. Data presented is the mean of three different experiments.
3.6. FT-IR analysis The synthesized nanoparticle was dried and subjected to FT-IR studies in order to determine the type of responsible biomolecules that aided the synthesized AgNPs. Fig. 6 shows the FTIR spectra of AgNPs synthesized using purified URAK. Two peaks at 1039 and 1458 which are the stretches due to C–O–C & C–OH and C C stretching respectively which corroborates with earlier reports [26,27]. Therefore, it can be confirmed that proteins are responsible for reduction of Ag+ ions. In the case of AuNPs synthesis FT-IR analysis showed two peaks at 1025, 1220, 1622, 1738 which corresponds to the C–N stretching in aliphatic amines, amide III and Amide I bands of proteins and carbonyl groups respectively [28] (data not shown). 3.7. Cytotoxic analysis of URAK In order to determine the effect of different concentration of URAK on the viability of cells, macrophage (RAW 264.7) was used. In all the concentration tested, the viability of macrophages, URAK did not decrease viability in any of the concentration tested (10 ng/ml–100 g/ml) significantly. 4. Discussion This manuscript describes the use of URAK in synthesizing AgNPs and AuNPs. URAK is a fibrinolytic enzyme isolated and purified from the culture supernatant of B. cereus NK1. The enzyme was produced in the optimized medium and purified as reported. The fractions that contained the fibrinolytic activity and homogeneity in SDS-PAGE were pooled and used for further experiments [20]. To the best of our knowledge this is the first report claiming the synthesis of both the nanoparticles by a single enzyme. Previously biosynthesis of nanoparticles has been reported by various organisms and plants including F. oxysporum [17], B. casei [16], Magnolia leaf broth [32] etc. This method was previously considered to be advantageous as they prevented the use of various toxic chemical reducing agents like sodium borohydride (NaBH4 ) and hydrazine (NH2 –NH2 ) These extremely reactive chemicals does not only possess environmental but also biological risks too [33]. But, microbial synthesis also has the possibility of presence of toxic substances like endotoxin on the surface of the nanoparticles. The contaminants present with the nanoparticle may hinder the use of nanoparticles in various biological applications, which requires the synthesized nanoparticle to be highly purified from the immunomodulating contaminants present [19]. Synthesis of NPs from culture supernatants may be little more advantageous
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than using the biomass, but the problem arises during the purification of the particles from the other molecules present on the culture supernatant [17]. Since macrophages are one of the immune cells first encounter the foreign materials the effect of URAK was checked in it and the enzyme did not induce toxicity up to the concentration of 100 g/ml. Therefore synthesis of nanoparticles by specific enzyme which is non toxic to the mammalian cells is highly advantageous and this would greatly reduce the downstream processing steps in obtaining pure nanoparticles for various applications. Moreover, the physical and chemical methods for the production at largescale usually results in larger particles whereas the biosynthesis can be effectively used for the production of smaller nanoparticles at large-scales operations [31]. Purification of nanoparticles from the contaminations remains a great problem during biosynthesis. Hence, we report here the synthesis of nanoparticles using eco-friendly methods, which may prevent the contamination of the environment by both the toxic chemicals and other biological substances. Here, purified URAK also act as a capping agent too which also prevents the agglomeration of the synthesized AgNPs and AuNPs. In chemical synthesis this process is an additional step. When bacteria or other biomass are used for the synthesis of nanoparticles, most of the particle adhere to the biomass itself and great efforts have to be put forth for the down stream processing steps [15]. If not during disposal into the environment the synthesized nanoparticles may accumulate in the environment affecting the microbial diversity, as AgNPs were shown to have potential anti-microbial activity and AuNPs are relatively costlier. Since we have used purified URAK without the addition of any other agents it can be concluded that URAK has the ability to synthesize AgNPs and AuNPs. Previously nitrate reductase mediated AgNPs synthesis resulted in the nanoparticle size range of 10–25 nm and a capping peptide is added to stabilize the synthesized nanoparticles [21]. Besides nitrate reductase, Bovine serum albumin and various extracellular fractions have been reported to synthesize AgNPs [17,29]. Some of the previous reports have shown that increasing the alkalinity of the reaction mixture, the formation of AgNPs is rapid [16]. During our experiment also increasing the alkalinity resulted in faster synthesis of nanoparticles. Similarly, AuNPs have been previously synthesized using the enzyme ␣-amylase and the activity of ␣-amylase was not affected after the synthesis of nanoparticles [22]. Protease enzyme from Actinobacter sp. has been reported to aid the synthesis of AuNPs [30]. However, BSA did not aid the synthesis of AuNPs when incubated with HAuCl4 alone [29]. Here the pure enzyme URAK aided the synthesis of both AuNPs and AgNPs which was also found to bind with the nanoparticle synthesized, becoming a stabilizer. Moreover, when nanoparticles synthesized using NaOH free mixture was analyzed, it retained the activity of the enzyme. Primarily the synthesis was characterized by UV–visible spectroscopic measurements. This corresponds with the previous reports where AgNPs and AuNPs were shown to have peak intensities in the range of 440 nm [16,23] and 540 nm [24] respectively. Moreover, increasing the pH enhanced the rate of synthesis of nanoparticle synthesis. This greatly corroborates with the previous conditions that alkaline conditions favors the synthesis of silver nanoparticles [10,25]. Presence of OH− ions reduced the time for AgNPs and AuNPs synthesized, whereas when the concentration of hydroxide ion was lesser, the time taken for the synthesis of AgNPs and AuNPs was very much longer. This shows that addition of OH− ions factious the nucleation resulting in the rapid synthesis of nanoparticles [16]. Although nanoparticles are bound to the enzyme, the presence of amino acids, especially tryptophan and tyrosine are previously reported to possess capacity to reduce various metal ions [29]. A
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careful genome analysis showed that B. cereus contains a gene for alkaline serine protease, that codes for protein with a length of 397 amino acids (GI: 225785743). The approximate molecular weight calculated from the sequence is found to be 43 kDa, which is very close to the obtained molecular weight of the purified URAK from B. cereus NK1. The amino acid sequence present in the protein contains 5 tryptophan residues and 23 tyrosine residues. These amino acids not only reduce the metal ions but also reported to cap the metal nanoparticles formed. Besides synthesis of nanoparticles, in NF mixture, both the particles contained enzymes with very good activity. Both the AuNPs and AgNPs synthesized had been checked for fibrinolytic activity and it was present. This was compared with biologically synthesized nanoparticles which did not exhibit any activity. Therefore, besides synthesis of AuNPs and AgNPs, the protein was found to bind on them and both the nanoparticles were checked for thermo and pH stability. Both the nanoparticles enhanced the stability of the immobilized protein. Therefore this might be an easy method for immobilization of fibrinolytic URAK. Previous methods have used a linker to immobilize the protein where the nanoparticles were synthesized separately, using chemical methods and linked with the protein. Here the nanoparticles were synthesized and URAK was immobilized with relative ease. Previously, various enzymes have been immobilized on both AuNPs and AgNPs. One such enzyme is Glucose oxidase. Glucose oxidase has been immobilized both on AuNPs and AgNPs with different linkers like carbodiimide linker and 6-aminohexanoic acid for the purpose of biosensor fabrication. Both the methods have been found to enhance the stability of the enzyme [34,35]. For immobilization of glucose oxidase, the nanoparticles were synthesized separately, purified enzyme separately and linked by linkers. But with URAK, no linkers have been used and the enzyme itself synthesized nanoparticles. 5. Conclusion The reduction of Ag+ and Au3+ ions by purified URAK has been reported here. The reduction resulted in the formation of AgNPs and AuNPs of spherical morphology. Here URAK not only synthesized nanoparticles but it remained bound with the nanoparticles as a capping agent thus preventing the agglomeration of the nanoparticles. This method may be highly useful as this method can be easily scaled-up and the eco-friendly nature of the nanoparticle synthesis methodology is one of the advantages. Moreover, the yield obtained can be the maximum as pure nanoparticles are present without any contaminants. Acknowledgements The author V. Deepak gratefully acknowledges Council for Scientific and Industrial Research (CSIR), India for providing Senior Research Fellowship (reference number: 9/1012(0001)/2k10-EMRI). We also thank Prof. Pushpa Viswanathan, Cancer Institute (WIA),
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