Size-Dependent Synthesis of Gold Nanoparticles and Their

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Size-dependent synthesis of gold nanoparticles and its peroxidase-like activity for the colorimetric detection of glutathione from human blood serum Vijay Kumar, Daraksha Bano, Devendra Kumar Singh, Sweta Mohan, Vikas Kumar Singh, and Syed Hadi Hasan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00503 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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ACS Sustainable Chemistry & Engineering

Size-dependent synthesis of gold nanoparticles and its peroxidase-like activity for the colorimetric detection of glutathione from human blood serum Vijay Kumar1, Daraksha Bano1, Devendra K. Singh1, Sweta Mohan1, Vikas Kumar Singh1 Syed Hadi Hasan1* 1

Nano Material Research Laboratory, Department of Chemistry, Indian Institute of Technology (BHU), Varanasi -221005, U.P., India.

*Corresponding author’s details: E-mail; [email protected], [email protected] Phone No.: +91-542-6702861 Mobile No.: +91 9839089919

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Abstract An eco-friendly and economic efficient approach has been developed for the photoinduced synthesis of more stable AuNPs using an aqueous extract of Croton bonplandianum (AEC) as a reducing and capping agent. The reaction mixture of AEC and HAuCl4.xH2O, when exposed to sunlight turned purple which primarily confirmed the biosynthesis of AuNPs. The biosynthesis was monitored using UV–vis spectroscopy which exhibited a sharp SPR band at 530 nm after 16 min of sunlight exposure. The parameters affecting the synthesis of AuNPs such as sunlight exposure, AEC inoculum dose and HAuCl4.xH2O concentration were also optimized. The HR-TEM study revealed that as the metal ion concentrations increased, the average size and anisotropic nature of the AuNPs increased. The X-ray diffraction pattern of AuNPs synthesized confirmed the formation of face-centered cubic crystal lattice of metallic gold. The involvement of polyphenolics in the synthesis of AuNPs was confirmed by comparing the FTIR analysis of pure tannic acid, AEC, pre and postannealed AuNPs. The XPS analysis corroborated the presence of two individual peaks attributing to the Au 4f7/2 and Au 4f5/2 binding energies which corresponded to the presence of metallic gold. The AuNPs thus obtained showed peroxidase-like mimicking activity which catalyzed the oxidation of TMB to oxTMB with the development of blue color and absorption spectra at 652 nm. However, the presence of GSH caused further reduction of oxTMB. This detection experiment showed an excellent linear relationship between 1 µM to 40 µM with the limit of detection 0.013 µM. In addition to this, the significant recovery of GSH from human blood serum advocated that the developed system was simple and sensitive for the real sample analysis. Keywords: Green synthesis; AuNPs; TEM; XPS; TMB; Peroxidase-like activity; Glutathione


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Introduction Glutathione (GSH), a thiol-containing tripeptide (Glu-Cys-Gly) is most abundant intracellular non-protein thiol present in plants, mammals, fungi and some prokaryotes. This thiolated tripeptide present in tissues exists in both the reduced form (GSH) and oxidized dimeric form (GSSH) which led it to maintain the intracellular reduction-oxidation potential. Apart from this, GSH plays a crucial biological role in cellular defense against xenobiotics, free radicals signal transduction, and gene regulation.1-3 The concentration of GSH ranged from millimolar (cells of tissues and organs) to micromolar (plasma, saliva, cerebrospinal fluid, urine) which changes dramatically under oxidative stress.4 The imbalance of cellular GSH leads to several diseases, such as aging, cancer, liver damage, leucocytes loss, psoriasis, liver damage heart problems and others.5-8 Therefore, there is an urgent need to develop a novel and efficient technique to monitor the changes in the concentration of GSH in biological fluids. Although various analytical methods such as chromatography9,10 photometry11, mass spectrometry12 and immunoiluminescence13 have been developed for the determination of GSH but all these methods have several disadvantages such as higher cost, low sensitivity and tedious pretreatment along with requirement of sophisticated instrumentation and skilled handling.14 Nano-biotechnology is an emerging field of science and technology for the development, improvement and utility of nanostructures. Since last two decades, the green synthesis of gold nanoparticles (AuNPs) has received the considerable attention of the researchers due to growing need in various fields; such as catalysis,15 biosensing,16 biological,17 electronics, photonics, drug delivery, biolabelling,18 wound dressings,19 and medicine.20 At present, plant extracts mediated green synthesis of AuNPs has proven to be more valuable than other biological systems because of avoiding the aseptic conditions and maintenance of microbial culture.21,22 Moreover, the plant 3 ACS Paragon Plus Environment


extract mediated synthesis is economically efficient and environment-friendly.23 The plant extracts are reported to contain several phytochemicals such as tannins, flavonoids, terpenoids, proteins, hydrogenases, and reductases, etc. which are responsible for both reduction and stabilization of AuNPs.21 Hence, currently, the plant extracts are being extensively used as a reducing and stabilizing agent for the synthesis of AuNPs. Recently, several plants like Pogestemon benghalensis,24 Avera lanata,25 Stevia reboundiana,26 and Eucalyptus oleosa27 have been used for the synthesis of AuNPs. At present the photoinduced green synthesis of AuNPs using plant extracts have become more economically efficient and eco-friendly where the rate of biosynthesis is increased by visible light.28 There are several articles which have been published for the biosynthesis of AuNPs using the sunlight induced route. In our previous study, we have reported the photoinduced synthesis of AgNPs using an aqueous extract of Croton bonplandianum,21 Erigeron bonariensis29 Xanthium strumarium30 and Murraya koenigi23 and Physalis angulata31 through sunlight induced route. The plant Croton bonplandianum, (Three-Leaved Caper) belongs to family Euphorbiaceae which is a native weed in Southwestern Brazil, Northern Argentina, Southern Bolivia, and Paraguay. In India, it is commonly found along the roads, canals and other waterstressed areas as an exotic weed. It is a medicinally important plant which has a great composition of reducing as well as capping agent required for the potent biosynthesis of AuNPs.32,33 Previously, we have reported the green synthesis of AgNPs using the extract of the same plant which showed that 30 min sunlight exposure time, 5.0% AEC dose and 4 mM AgNO3 concentration were the optimum parameters. The average size of the AgNPs obtained from the optimum AgNO3 concentration was 19.4 nm. The AgNPs thus obtained also showed selective 4 ACS Paragon Plus Environment

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detection of Fe (III) and potent antibacterial activity against both Gram-negative and Grampositive bacteria. Whereas the AuNPs synthesized in the current investigation showed that 24 min of sunlight exposure time, 4.0% of AEC dose and 1.6 mM HAuCl4.xH2O concentration were optimum conditions for the biosynthesis of AuNPs and the average size of the optimum HAuCl4.xH2O concentration was 8.6 nm. The AuNPs thus obtained showed peroxidase-like mimetic activity which was utilized for the colorimetric detection of glutathione. The main objective of the current investigation is to apply the principles of green chemistry for the development of a green, simple and eco-friendly route for the synthesis of stable AuNPs by applying the principles of green chemistry and by optimizing several process parameters using one parameter at a time approach. In addition to this, it was aimed at not to use any hazardous chemical, sophisticated instrumentation, heating and stirring too throughout the synthesis of AuNPs. The investigation of the effect of different metal ion concentrations on the shape and size of AuNPs and crystallinity was one of the important parts of our aim. Finally, the optimum AuNPs thus obtained was investigated for its peroxidase-like activity towards the oxidation of TMB with the development of blue color which was considered as a base for the detection of GSH. Materials and Method The chemical Chloroauric acid (AuCl4.xH2O) used as a precursor for biosynthesis of AuNPs was procured from Loba Chemicals. Tetramethylbenzidine (TMB) and Hydrogen Peroxide (H2O2) were procured from Merck, India. Ethanol was procured from Avra Chemical Lmtd, India. The amino acids like glycine (Gly), alanine (Ala), valine (Val), proline (Pro), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), glutathione (GSH) and other 11 type



amino acids were brought form Loba Chemicals. Fresh leaves of C. bonplandianum were collected from campus area of Indian Institute of Technology (Banaras Hindu University), Varanasi, India and which were further brought and processed in the lab to prepare leaf extract. All the reagents used in the experiments were of analytical grade and used as received from the source. Preparation of leaf extract The aqueous leaf extract of C. bonplandianum was prepared according to our previous report

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. Briefly, the collected fresh leaves were first washed under tap water and then several

times using deionized water to eradicate dust and others adhering impurities properly. After that, the leaves were air dried to remove the moisture. Further, the dried leaves were cut into small pieces, and 12.5 g of it was heated for 10 min in 50 mL of deionized water at 80 °C. Then, the aqueous extract of C. bonplandianum (AEC) was collected and filtered using Whatman filter paper No. 1. Thus prepared AEC was stored at 4°C as a stock solution and used within three days (Scheme Supporting Information 1). After preparation, the presence of polyphenolics was confirmed through ferric chloride test by adding 0.1% FeCl3 solution into the AEC (Scheme Supporting Information 2). Further, the quantification of total phenolic was investigated through Folin-Ciocalteu’s method. Briefly, 1 mL of plant extracts or standard solutions of prepared gallic acid were added in test tube which is followed by the addition of 1 mL of Folin Ciocalteu’s reagent. After 5 minutes, 1.0 mL of 7 % Na2CO3 was added to this, and total volume was made up to 10 mL. After 90 min, the absorbance was determined against reagent blank at 650 nm (Fig. Supporting Information 1). Biosynthesis of AuNPs


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The biosynthesis reaction of AuNPs was carried out in both natural sunlight and dark condition to investigate the efficacy of photocatalytic activity of the sunlight. The temperature of the ambient environment in sunlight and solar intensity of incident sunlight radiation were 38°C and 63600 lux respectively. The temperature of the dark condition and light intensity was 32°C and 0 lux respectively. The synthesis reactions were carried out between 12 pm to 2 pm to avoid the fluctuation in temperature and sunlight intensity. For the synthesis purpose, 2% (v/v) of AEC inoculum dose was added into two mL of 0.8 mM AuCl4.xH2O solution at neutral pH and kept in both bright sunlight and dark condition. The reaction mixtures keeping in sunlight exhibited a quick change in color within 16 min from light yellow to dark purple whereas the reaction mixtures kept in the dark did not show the same degree of color change even after 10 hrs (Fig. Supporting Information 2). Therefore, further, all the AuNPs synthesis experiments were performed in sunlight to optimize the other process parameters using one factor at a time approach. The process parameters such as sunlight exposure time, AEC inoculum doses and AuCl4.xH2O concentrations were screened in the range of 4 to 24 min, 1.0% to 8.0% (v/v) and from 0.4 mM to 3.2 mM respectively. After the optimization process, the AuNPs thus obtained were purified. For this, the AuNPs were firstly centrifuged at 15000 rpm for 15 min and subsequently re-dispersed in deionized water to remove the water-soluble molecules and other secondary metabolites. The final mass of AuNPs was collected by vacuum drying after repeating this process four times. Detection of glutathione Firstly, the various solutions of the different concentration of GSH were prepared by serial dilution of the prepared stock solution of GSH. The detection of the GSH was carried out by adding 50 µL TMB (1 mM), and 50 µL H2O2 (1 mM) into 200 µL of 0.2 M NaAc buffer (pH 7 ACS Paragon Plus Environment


4) then 50 µL AuNPs. After that, the reaction mixture was incubated in the dark for 10 min at room temperature (32 ºC) which was followed by adding the different concentration of GSH and measured through UV-vis spectrophotometer. To examine the selectivity of the GSH, 50 µL of (100 µM) of each Gly, Val, Ala, Leu, Ile, Phe, Ser, Thr, Tyr, Met, His, Asp, Glu, Trp, Lys, Arg, Asn, Pro, Cys, and GSH were introduced into the AuNPs+TMB+H2O2 system. After that, the samples were incubated for 10 min at room temperature and further preceded by measuring the absorption spectra. For obtaining the detectable linear range of the GSH, the sensitivity was measured in the range from 1 µM to 120 µM. Characterization of AuNPs. The AuNPs thus obtained were characterized through various instrumental techniques empowering the nanotechnology. The primary confirmation study of AuNPs was performed using UV–visible spectrophotometer (Evolution 201, Thermo Scientific) in the range of 300 to 800 nm. The important roles played by the functional groups in the synthesis and stabilization were studied by Fourier Transform Infrared Spectrophotometer (FTIR, Perkin Elmer Spectrum 100) in the range of 4000–400 cm-1. The crystallinity of AuNPs was investigated using X-ray Diffractometer (Rigaku Miniflex II) having Cu Kα radiation source and Ni filter in the range of 20° to 80° at the scanning rate of 6° min−1 with a step size 0.02°. The shape and size of the AuNPs were further confirmed by Transmission Electron Microscopy (TEM) carried out on TECNAI 20 G2-electron microscope operated at accelerating voltage 200 kV. The purity and elemental composition of AuNPs were confirmed by EDX analysis equipped with TEM. The Selected Area Electron Diffraction (SAED) demonstrated the presence of several concentric diffraction rings which also confirmed the crystallinity of synthesized AuNPs. The speciation of 8 ACS Paragon Plus Environment

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biosynthesized AuNPs was confirmed by X-Ray Photoelectron Spectroscopy (XPS, AMICUS, Kratos Analytical, A Shimadzu) with Mg Kα (1253.6 eV) radiation as an X-ray source. Zetapotential of the AuNPs was measured using a Malvern Zetasizer (Malvern Instruments, Ltd.). Results and Discussion Primary confirmation The surface plasmon resonance (SPR) which is produced through collective oscillations of free conduction electrons caused to change in color of the reaction mixture from yellowish to purple. This change in color was the preliminary confirmation of biosynthesis of AuNPs. The reaction mixture exposed to bright sunlight exhibited the development of dark purple color and sharp SPR band within 16 min which indicated the rapid synthesis of AuNPs. Whereas the reaction mixture kept at the darker condition did not reveal the characteristics purple color even after 10 hrs of incubation (Fig. Supporting Information 2). This time lag indicated that reaction mixture exposed to sunlight corroborated rapid biosynthesis of AuNPs as compared to reaction mixture kept at darker condition. The UV–visible absorption spectroscopy is an important tool to monitor the formation AuNPs. The effect of bright sunlight exposure on the synthesis of AuNPs, color change and the pattern of SPR band was studied by withdrawing the samples from the reaction mixture at a regular time interval of 4 min and screened between 300 to 800 nm through UV-visible spectroscopy. It was found that after 16 min of bright sunlight exposure, the screened sample showed an SPR band at 542 nm which attributed to the characteristic surface plasmon resonance of AuNPs having λmax values in the range of 500-600 nm.34 Optimization of the synthesis process Sunlight exposure



The optimization of sunlight exposure time for the synthesis of AuNPs was carried out by screening the reaction mixtures at each 4 min interval of time. It was noticed that as the exposure time increased, the pattern of change in color from light yellow to dark purple of each AEC doses increased (Fig. 1A-H). The intensity and color of the reaction mixture of first 8 min and 4 min of exposure of 1% and 2% AEC respectively did not show characteristics SPR band and change in color. The above phenomenon was due to the absence of the nuclei with the radius larger than critical radius which re-dissolved in the reaction mixture and failed to nucleate to form AuNPs (Fig. 1A and B).35,36 After that, the intensity of the SPR band continued to be increased up to 24 min of each inoculums dose by 4% AEC and after that no significant increase in SPR band intensity was observed ((Fig. 1A-D). The increase in intensity signified the increased synthesis of AuNPs whereas further, no increase in SPR band intensity confirmed the establishment of the equilibrium.31 After that, an interesting result was obtained while screening the SPR band pattern of 5% to 8% AEC. After achieving the maximum intensity with the proceeding of exposure time, a regular pattern of decrease in SPR band intensity was observed with the increase in AEC doses from 5% to 8%. This interesting pattern was due to the increased agglomeration and accordingly decreased SPR band intensity with the time by increasing AEC doses (Fig. 1E-H). AEC Inoculum dose Figure 1A-H represented the UV-visible spectra of AuNPs produced by a different stock solution of AEC from 1.0% to 8.0%. While screening it was observed that as the AEC dose increased from 1% to 8%, the color intensity of the reaction mixture increased from light purple to dark purple at each time interval which indicated the increased synthesis of AuNPs.30 The pattern of UV-visible spectra revealed that with the increase in stock solution, the intensity of the 10 ACS Paragon Plus Environment

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SPR band increased up to 4 %. After that, a decreasing pattern of SPR band was observed after each 4 min of exposure time. It was noticed that the exposure time required for the development of the SPR band and the color of the solution decreased with the increasing amount of the AEC stock solution (Fig. 1A-H). When 1% AEC was screened, a single SPR was produced with broader and less intense peak which suggested that the synthesized AuNPs were fewer in number. Further, on increasing the AEC dose up to 4.0 %, the intensity of the SPR band increased up to 24 min exposure time which indicated the increased synthesis of AuNPs with increasing the AEC dose.21 Thereafter, by using 5%, 6%, 7% and 8% AEC, a gradual decrease in SPR band intensity was observed. The decreasing of the sharpness and increase in broadening of the SPR band towards longer wavelength was due to the overgrowth of the AuNPs which got lighter in color due to settling at the bottom that can be seen very clearly from the attached vial color of 8% AEC after 28 min exposure time. Therefore 4% AEC was considered as optimum for the synthesis of AuNPs. HAuCl4.xH2O concentration The synthesis of AuNPs was also optimized using various concentration of HAuCl4.xH2O from 0.4 mM to 3.2 mM at 4% AEC stock solution and 24 min of sunlight exposure time. The SPR bands of 0.4 mM, 0.8 mM, 1.2 mM, 1.6 mM, 2.0 mM, 2.4 mM, 2.8 mM and 3.2 mM HAuCl4.xH2O concentrations revealed the maximum absorbance at 560 nm, 556 nm, 550 nm, 552 nm, 545 nm and 543 nm, 546 nm and 595 nm respectively after 24 min of sunlight exposure which is shown in Figure 2A-H. The figures indicated that as the concentration of each HAuCl4.xH2O from 0.4 mM to 3.2 mM increased, the color of reaction mixtures darkened gradually from 4 to 24 min. It is well known that the color of AuNPs solution is associated with the size of nanoparticles which was confirmed by TEM analysis. It was observed that the 11 ACS Paragon Plus Environment


average size of the AuNPs increased from 5.6 nm to 19.4 nm with the increase in HAuCl4.xH2O concentration from 0.4 mM to 3.2 mM (Fig. 2A-H). Thus it is clear that the color governs the particle size distribution.37 When the concentrations of HAuCl4.xH2O were increased from 0.4 mM to 3.2 mM, a distinct pattern of SPR bands were observed. It was observed that while using 0.4 mM HAuCl4.xH2O concentration, the SPR bands obtained were less intense and the color of the solution was also very light which continued to be increased at each time interval from 4-24 min. This indicated the synthesis of very few numbers of AuNPs with smaller size which was also confirmed by the produced TEM image (Fig. 5A). This pattern of increasing SPR band intensity with time was continued up to 1.6 mM HAuCl4.xH2O concentration. The increase in SPR band intensity up to 1.6 mM at each screening time interval indicated that as HAuCl4.xH2O concentration increased, the synthesis of AuNPs also increased with increased average size which was further confirmed by TEM images (Fig. 5B-D).38 But when the HAuCl4.xH2O concentration increased beyond 1.6 mM, the SPR band produced were broader and steeper towards longer wavelength with continued decreasing intensity up to 3.2 mM. This indicated the synthesis of larger AuNPs which might be due to overgrowth as can be seen from the TEM images (Figure 5E-H). It is well reported fact that spherical/pseudospherical nanoparticles are formed when the generated nuclei having low concentration of precursor monomers. However, the anisotropic nanoparticles are formed when the generated nuclei having high concentration of precursor monomers. It was observed that on increasing the HAuCl4.xH2O concentration, the formation of anisotropic AuNPs like trigonal, rod, hexagonal, pentagonal-shaped increased due to increased monomer concentration which is obvious from the results shown in Figure 5E-H.39


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Therefore, to get smaller and large number of AuNPs with controlled growth and smaller size, 1.6 mM HAuCl4.xH2O concentration at 4.0% AEC and 24 min exposure time was chosen as the optimal condition for the current study. Mechanism involving AuNPs formation The presence of polyphenolic compounds (tannin, flavonoids) resins, sugars, proteins, alkaloids, glycosides, and steroids have already reported by several authors through the phytochemical analysis of C. bonplanadianum leaf extract.40 Flavonoids, tannins, and sugars act as a reducing agent whereas proteins and some other phytochemicals act as a stabilizing agent during synthesis of AuNPs.28 The previous phytochemical studies of C. bonplanadianum, our ferric chloride test and Folin Ciocalteu’s method, photoinduced synthesis of AuNPs and FTIR analysis of AEC, TA, pre-annealed AuNPs and post-annealed AuNPs encouraged us to propose a mechanism of AuNPs synthesis through AEC. The presence of a vast variety of phytochemicals in the AEC may fallow different reduction routes of Au3+, but the presence of tannins (polyphenol) in AEC was comparatively higher than other chemicals. Therefore, the reduction of Au3+ was proposed using tannin as a reducing agent. The authors have proposed a stepwise mechanism of AuNPs synthesis. When the AEC was added to the HAuCl4.xH2O solution, the OH group of polyphenolic compound (tannin) present in AEC bound with Au3+ and formed the Au3+/AEC complex. Since the results showed that the biosynthesis of AuNPs was driven by the photocatalytic effect of sunlight. The photoactivation of Au3+/AEC complex was the first step of the AuNPs synthesis where the OH group of AEC produced hydrated electrons which put strong evidence after the sunlight induced synthesis of AuNPs.41,42 The second step involved the reduction of Au3+ to Au0 by the hydrated electrons produced earlier.43 The concentration of gold atoms (Au0) continued to increase with 13 ACS Paragon Plus Environment


the continued reduction of HAuCl4.xH2O. When the increased concentration of Au0 atoms exceeded the critical supersaturation, the gold atoms started to nucleate and formed the crystal nuclei (step III). The formation of these crystal nuclei led to the decrease in concentration of Au0 atoms below to the critical supersaturation where the whole process is dominated by the growth of nuclei to form nanoclusters because of no longer increase in the number of crystal nuclei (step IV). The growth of the nanoclusters further decreased the concentration of Au0 atoms below the saturation level which stopped the growth of nanoclusters and finally aggregated to form AuNPs (step V)44 (Scheme 1). The photoresponsive nature of the polyphenolics and others flavin binding protein present in AEC further advocated the photocatalytic action of sunlight where the electrons could be easily donated by the polyphenolics in the presence of the sunlight or can stimulate other phytochemicals to do the same.45 In this case proteins, sugars or some other phytochemicals act as stabilizers for AuNPs. The FTIR also advocated the above fact by illustrating the contribution of –OH and –NH2 groups in synthesis and stabilization of AuNPs. Scheme 2 showed the photoactivation of Au3+/AEC complex (step I), reduction of Au3+ to Au0 by involving n(OH) of n(tannin) present in AEC (step II), nucleation (step III), to form nanocluster (step IV) and finally AuNPs by overgrowth (step V). Characterization Thus synthesized optimum AuNPs at 24 min of sun light exposure time, 1.6 mM HAuCl4.xH2O concentration and 4.0% of AEC dose were investigated through various modern characterizing techniques such as FTIR, XRD, TEM, SAED, EDX, and XPS analysis. The phytochemicals responsible for the biosynthesis and stabilization of AuNPs were explored by FTIR study. On the basis of the phytochemical profile of the leaf extract (Fig. 14 ACS Paragon Plus Environment

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Supporting Information 1), it was concluded that the polyphenolic compounds like tannin were the main constituents of the AEC. To strengthen this fact, the FTIR analysis of the pure TA was also carried out and matched with that of AEC. Figure 3 represented the FTIR spectra of pure TA, AEC, pre-annealed AuNPs and post-annealed AuNPs. The FTIR spectrum of pure TA was well matched with the FTIR spectra of AEC to confirm the presence of specific functional groups of tannin present in AEC. The FTIR spectrum of AEC was taken as a control and compared with the FTIR spectra of pre-annealed AuNPs to identify major functional groups responsible for the biosynthesis of the AuNPs. The phytochemicals involved in the capping of the AuNPs was also confirmed by comparing the FTIR analysis of post-annealed AuNPs with the FTIR analysis of pre-annealed AuNPs. It was found that the FTIR spectrum of the pure (tannic acid) TA showed the characteristics band at 3339 cm-1 (ʋs of OH), 2939 cm-1 (ʋs of C=C-H), 1711 cm-1 (ʋs of CO), 1616 cm-1 (ʋs of C=C), 1449 cm-1 (ʋs of C=C ), 1340 cm-1 (ʋs of C=C) 1020 cm-1(ʋs of O-C) and 620 cm-1(ʋb, bending vibration)21. When the FTIR spectrum of the AEC was carried out, it was noticed that it showed the similar band as shown by TA. The presence of similar characteristics band both in TA and in AEC confirmed the presence of tannin like moieties (polyphenolic). The presence of tannin in AEC was also strengthened by carrying out an experiment which instantly turned the greenish-yellow color of the AEC to dark green color after adding 0.1% of FeCl3 solution (Fig. Supporting Information 2).46 The FTIR spectrum of the pre-annealed AuNPs showed the same characteristic bands as that of AEC except some diminishing and shifting which advocated the significant role of phytochemicals present in AEC involved in the synthesis and capping of the AuNPs. Both the spectra showed a shifting in following peaks: from 3359 to 3415 cm-1 (band due to ʋs of OH of the polyphenolic compounds and ʋs of NH of the proteins), 15 ACS Paragon Plus Environment


1051 cm-1 to 1027 cm-1 (ʋs of O-C).47,48 The peak present at 2929 cm-1 (ʋs of C=C-H), 1640 cm-1 (ʋs of C=C), and 1318 cm-1 (ʋs of C=C) did not show any shift rather diminishing whereas the peak present at 610 cm-1 completely disappeared. These investigations revealed the important role played in the biosynthesis of AuNPs by the hydroxyl, carboxyl, amino and amide groups of phytochemicals present in AEC. The tannins, glucose, and protein present in AEC could be the major source of these groups. The FTIR spectra of post-annealed AuNPs showed decrease in the intensity of the characteristics peak which confirmed the loss of phytochemicals involved in the capping of AuNPs. The crystalline nature of the AEC synthesized AuNPs of all the metal ion concentrations (0.4 to 3.2 mM) was determined by XRD analysis. The data were collected in the angular range 30° ≤ 2θ ≤ 80° with step size 0.02° and scan rate 6° min-1.The characteristic XRD patterns of AuNPs are represented in Figure Supporting Information 3 with the diffraction peaks observed at 2θ = 38.06º, 44.24°, 64.47° and 77.3°. The peaks of AuNPs synthesized from 0.4 to 3.2 mM were well matched with standard diffraction data with those reported for gold by Joint Committee on Powder Diffraction Standards (JCPDS) file no: 040784 and attributed to (111), (200), (220) and (311) Bragg reflections respectively. These Bragg reflections corresponded to the crystalline planes of the face-centered cubic (fcc) crystal lattice of metallic gold. The average estimated crystallite size of the all the metal ion concentrations (0.4 to 3.2 mM) AuNPs were consistent with the average sizes of AuNPs obtained from the HR-TEM analysis (Table Supporting Information 1). The exact size and morphology of the AuNPs was analyzed using the different concentration of AuCl4.xH2O (0.4 mM to 3.2 mM) and was investigated through HR-TEM analysis. For the HR-TEM analysis, AuNPs suspension obtained from all the AuCl4.xH2O (0.4 16 ACS Paragon Plus Environment

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mM to 3.2 mM) concentrations were first diluted by DW (1:3). Further, the carbon-coated copper grids were dipped into respective AuNPs solutions and then dried under table lamp for two hr and loaded onto a specimen holder. The HR-TEM images of AuNPs with different magnifications produced from 0.4 mM to 3.2 mM are shown in Figure 4 A-H. The HR-TEM images obtained from 0.4 mM revealed the presence of smaller and spherical AuNPs. The images at different magnification also revealed that there was very less number of particles distributed throughout the mass of the samples which was in accordance with the change in color and SPR band intensity. The corresponding size distribution histogram of AuNPs represented that maximum AuNPs were in the range of 2.5 nm to 4.5 nm having an average size distribution of 5.6 nm. Whereas when the concentration of the AuCl4.xH2O increased to 0.8 mM, the number of spherical shaped AuNPs increased. The corresponding size distribution histogram represented that the synthesized AuNPs were in the range of 1.4 nm to 11.8 nm with the average size 5.7 nm. When the concentration was further increased by 1.2 mM, the number of spherical AuNPs thus produced also increased and observed in the range of the 1 nm to 17 nm with the average size of 6.1 nm as revealed by histogram. It was noticed that as the concentration of AuCl4.xH2O increased by 1.6 mM, the formation of spherical AuNPs with somewhat larger size was maintained up to this. The histogram revealed that the AuNPs were in the range of 1 nm to 19 nm with an average size 8.6 nm. Further, the TEM analysis of 2.0 mM to 3.2 mM HAuCl3.xH2O concentrations revealed that the synthesis of anisotropic like trigonal, hexagonal AuNPs with larger size and range were increased. The anisotropic AuNPs produced from 2.0 mM, 2.4 mM, 2.8 mM, and 3.2 mM ranged from 1 nm to 37 nm, 4.5 nm to 52 nm, 6 nm to 60 nm, and 11 nm to 79 nm with an average size 13.2 nm, 14.5 nm, 17.68 nm, and 19.4 nm respectively. In brief, it was noticed that on increasing the HAuCl4.xH2O concentrations, the average size of the AuNPs



and the synthesis of larger anisotropic AuNPs increased (Fig. 5A). The pattern of increasing average size of AuNPs corroborated a good linear relationship by increasing the HAuCl4.xH2O concentrations having R2 = 0.976 (Fig. 5B). A close look at the SAED pattern of the 0.4 mM AuCl4.xH2O concentrations suggested very less circular ring which advocated the fewer number of AuNPs whereas others suggested the presence of clear circular rings advocating the crystalline nature of the AuNPs. The SAED pattern of all the AuNPs obtained from each AuCl4.xH2O concentrations showed the same circular ring pattern showing the crystalline nature of the AuNPs. The XPS analysis was used to characterize the elemental composition of the synthesized AuNPs. The survey scan spectrum of AuNPs is shown in Figure 6A. The wide scan survey spectrum exhibited the peaks corresponding to Au 4f, C 1s, and O 1s which depicted Au, C, and O as a major element present in the sample. The peaks of C and O indicated the involvement of biological moieties in the stabilization of the AuNPs. Figure 6B showed the Au 4f core level spectrum of AuNPs. The Au 4f spectrum of Au corroborated two peaks at 83.48 and 87.64 eV which corresponded to the binding energies of Au 4f7/2 and Au 4f5/2 respectively. The splitting of the 3d doublet of Au was 4.16 eV, indicating the formation of metallic AuNPs (Au0) which indicated to the presence of pure Au.49 The core level C 1s spectrum (Fig. 6C) had three Gaussian peaks at 286.9 and 288.4 eV and 289.7 eV which assigned to C–O–C/C–OH, C=O/C– Au and COOH/O–C=O respectively. Figure 6D which showed three peaks of the O 1s signal at 532.0, 533.1, 533.7 and 534.3 eV. The peak at 532.0 eV indicated the presence of, C=O whereas the peaks at 533.1 eV and 533.7 eV represented to the C–O and C–O–C. The peak observed at 534.3 eV advocated the presence of and C–OH respectively.


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The zeta potential study indicated that the surface of the AuNPs was negatively charged (-11.5 mV) which exhibited good stability in suspension (Fig. Supporting Information 4). Peroxidase-like catalytic activity of green synthesized AuNPs The peroxidase-like activity of the green synthesized AuNPs was investigated using the AuNPs+TMB+H2O2 system where TMB was taken as an indicator which produced blue color and intense spectra at 652 nm in oxidized form (oxTMB). When AuNPs was added to the TMB+H2O2 system, the color changed from colorless to blue color along with sharp spectra at 652 nm was observed. This phenomenon indicated the peroxidase-like activity of the green synthesized AuNPs which catalyzes the oxidation of TMB in the presence of H2O2 (Scheme 3). The above catalytic pathway of the oxidation of TMB can be monitored by measuring the change in absorbance at 652 nm. The addition of GSH to the AuNPs+TMB+H2O2 system resulted in a decrease in absorbance at 652 nm and change in color from blue to light blue and after that, almost colorless. This phenomenon may be due to the reduction of the oxTMB by GSH50 (Scheme 3). From the finding of such result, the authors were encouraged to establish a simple method for the selective and sensitive colorimetric detection of GSH. The UV-visible absorption spectra of TMB, H2O2, TMB+H2O2, AuNPs, and AuNPs+TMB+H2O2 and their respective color are presented in Figure Supporting Information 5A. It was observed that AuNPs produced the spectrum at 542 nm whereas TMB, H2O2, TMB+H2O2 neither produced any spectra nor any color. However, when AuNPs was added in TMB+H2O2, an intense spectrum at 652 nm with blue color was produced which indicated that green synthesized AuNPs can be used for the catalytic oxidation of TMB to oxTMB. Whereas when GSH was added to the AuNPs+TMB+H2O2, the blue color disappeared and the absorbance of the spectra decreased (Fig. Supporting Information 5B). 19 ACS Paragon Plus Environment


Optimization of the experimental factors The various factors affecting the peroxidase-like activity of AuNPs such as incubation time, pH, temp, TMB concentration, H2O2 concentration, AuNPs concentrations were optimized using one parameter at a time approach for obtaining the optimum result for the better performance against the detection of glutathione. Figure Supporting Information 6A showed the peroxidase-like activity of AuNPs at different pHs in the range of 1-10. It was observed that AuNPs exhibited excellent peroxidaselike activity at pH 4. As the pH increased beyond four the peroxidase-like activity of AuNPs started to decrease towards higher pH due to decomposition of H2O2 into H2O and O2 rather OH• radical.51 Therefore, pH four was considered as optimum pH for better peroxidase-like activity of AuNPs. After that, the effect of temperature on the peroxidase-like activity of AuNPs was also optimized at constant pH 4. Figure Supporting Information 6B showed the optimum peroxidase-like activity at temperature 40 ºC. Further, on increasing the temperature, the peroxidase-like activity decreased due to agglomeration of AuNPs which led to the hindrance of electron transfer.51 For optimizing the incubation time required for obtaining the maximum absorbance at 652 nm, the samples were screened, and UV-visible spectra were recorded simultaneously. Figure 7A showed the absorption spectra of the reaction system at 652 nm at different time interval. The darkening of the color from light blue to dark blue and intensity of the spectra were observed to be increased by increasing the incubation time only up to 18 min, and after that, no significant change was observed. Therefore, 18 min of incubation time was chosen as optimum time. The calibration plot of increase in absorbance vs. incubation time showed a good linear relationship in the range of 2-14 min with R2 = 0.975 (Fig. 7B). Similarly, the effect of different 20 ACS Paragon Plus Environment

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concentration of TMB, AuNPs amount and H2O2 concentration was optimized at 18 min of incubation time, pH 4, and temp 40 ºC. It was observed that initially, the peroxidase-like activity increased up to an optimum value. After that, no significant change in absorbance was observed. Figure 7C and D represented the optimum TMB concentration was 0.8 mM using 50 µL H2O2 (1 mM) and 50 µL AuNPs having a linear relationship between 0.1 mM to 0.8 mM with R2 = 0.993. Figure 8A and B showed that the optimum AuNPs amount was 80 µL using 50 µL H2O2 (1 mM) and 50 µL TMB (0.8 mM) having a linear relationship between 10 µL to 60 µL AuNPs with R2 = 0.976. The optimum activity of H2O2 was observed at 8 mM using 80 µL AuNPs and 50 µL TMB (0.8 mM) having a linear relationship between 2 mM to 8 mM with R2 = 0.997 (Fig. 8C and D). Beyond these optimum concentrations, no significant change in absorbance was observed the agglomeration of AuNPs due to the over concentration which increased Detection of glutathione The sensing potential of the proposed AuNPs+TMB+H2O2 system towards the detection of GSH was observed by a change in color of the system from blue color to colorless which can be seen by naked eye (Fig. Supporting Information 5B). It was observed that as the concentration of the GSH increased from 1.0 to 120 µM, the color of the AuNPs+TMB+H2O2 system turned dark blue to light blue due to the continued reduction of oxTMB into reduced form (GSSG) (Fig. 9A). The selectivity towards the colorimetric detection of GSH was examined by adding 50 µL (100 µM) of each Gly, Val, Ala, Leu, Ile, Phe, Ser, Thr, Tyr, Met, His, Asp, Glu, Trp, Lys, Arg, Asn, Pro, Cys, and GSH into the TMB+H2O2+AuNPs system. Figure Supporting Information 7 showed the ∆A of TMB+H2O2+AuNPs system at 652 nm after the introduction of GSH and various other amino acids. It was observed that ∆A of GSH was surprisingly larger 21 ACS Paragon Plus Environment


than others. A little change in absorbance (∆A) was observed with the Met and Cys which were negligible as compared to previously reported literature.52,53 Thus it was concluded that the developed sensor system showed good selectivity in the presence of others thiol-containing amino acids. Figure 9A revealed that no change in absorption at 652 nm and color was observed in the reaction system without GSH. Inset Figure 9A corroborated the gradual decrease in color intensity by increasing the GSH concentration up to 120 µM. It was also observed that when the concentration of the GSH increased from 1 to 120 µM, the absorbance at 652 nm decreased gradually due to the reduction of oxTMB.50 It can be concluded that the green synthesized AuNPs can be used as a green catalyst for the oxidation of TMB which can be used for the colorimetric detection of GSH. Figure 9B revealed a good linear relationship between absorbance and different concentration of the GSH from 1 µM to 40 µM with a high value of R2 (0.998). The limit of the detection (LOD) was calculated to be 0.013 µM which was better than previously reported works (Table 1). Detection in real samples High selectivity and sensitivity of the AuNPs+TMB+H2O2 system towards the detection of GSH encouraged us to detect the GSH in real samples. Hence; the utility of the AuNPs+TMB+H2O2 system was examined for the detection of GSH in human blood serum obtained from Sir Sundarlal Hospital, Banaras Hindu University, Varanasi, U.P., India, 221005. For this, the human serum samples were diluted and spiked with the known amount of GSH. Figure 9C and D showed the UV-visible spectra and bar diagram of serum samples at 652 nm. The spectra and bar diagram and Inset Figure indicated that serum 1 having highest concentration whereas serum 4 having the lowest concentration of GSH. The recovery of the 22 ACS Paragon Plus Environment

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GSH was calculated and given in Table 2. The recovered amount of GSH was in the range of 96.3% to 108.2%. Such a high recovery advocated that the AuNPs+TMB+H2O2 system is applicable and reliable for the real clinical samples analysis. Conclusions An attempt has been made to develop completely eco-friendly and economically viable route for the green synthesis of AuNPs. For this, AEC was used as a potent reducing and stabilizing agent. The photoinduced route was adopted as it avoided the need for heating and stirring. The parameters affecting the synthesis process were optimized using one parameter at a time approach and observed that 24 min of sunlight exposure time, 4% AEC inoculums dose and 1.6 mM HAuCl4.xH2O concentration were the optimum parameters. The comparative study of FTIR analysis of AEC with pure tannic acid efficiently confirmed the involvement of polyphenolics which were already reported and also confirmed by our group; in the synthesis and stabilization of AuNPs. The XRD analysis confirmed the synthesis of pure metallic gold with face centered cubic lattice. The TEM images revealed the increase in average size and anisotropy with the increase in HAuCl4.xH2O concentration. The presence of two individual peaks attributing to the binding energies of Au 4f7/2 and Au 4f5/2 in XPS analysis also strengthened the fact of the presence of metallic gold. The AuNPs thus obtained showed as a peroxidase-like mimicking activity which catalyzed the oxidation of TMB to oxTMB with the development of blue color and absorption spectra at 652 nm. The parameters affecting the peroxidase-like mimicking activity were also optimized where 18 min of incubation time, pH 4, 40 ºC of temp, 8 mM of H2O2 concentration, 80 µL of AuNPs amount, and 0.8 mM of TMB concentration were the optimum conditions. However, the presence of GSH caused further reduction of the oxTMB. This detection experiment showed the good linear relationship between 1 µM to 40 µM with the 23 ACS Paragon Plus Environment


limit of detection 0.013 µM. In addition to this, the greater recovery of GSH from human blood serum advocated that the developed system was simple and sensitive for the real sample analysis.


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Scheme Captions Scheme 1 Schematic representation of classical LaMer mechanism showing the concentration of monomer (Au0) during particle nucleation and growth. Scheme 2 The scheme showing the various steps of AuNPs synthesis by involving the enol form of ‘n’ number of polyphenolic groups of ‘n’ number of tannin present in AEC which formed complex (AEC/Au3+) with Au3+ and further reduced the n[Au3+] to n[Au0] by the hydrated electrons released from the debonding of O-H bond. The n[Au0] formed AuNPs after simultaneous nucleation, cluster formation and further growth. Scheme 3 The mechanism of peroxidase-like activity of AuNPs for the colorimetric detection of GSH.

Figure Captions: Figure 1 Monitoring of UV–vis absorption spectra of AuNPs synthesis using different AEC inoculums dose at 1% (A), 2% (B), 3% (C), 4.0% (D) 5% (E), 6% (F), 7% (G), and 8% (H); and their corresponding increase in intensity and color change pattern with the advancement of time from 4 to 28 min (conditions; 0.8 mM HAuCl4.xH2O concentration and 4 to 28 min sunlight exposure time). Figure 2 Monitoring of UV–vis absorption spectra of AuNPs synthesis using different HAuCl4.xH2O concentration at 0.4 mM (A), 0.8 mM (B), 1.2 mM (C), 1.6 mM (D) 2.0 mM (E), 2.4 mM (F), 2.8 mM (G), and 3.2 mM (H); and their corresponding increase in intensity and color change with the advancement of time from 4 to 24 min (conditions; 4% AEC inoculums dose and 4 to 28 min sunlight exposure time).



Figure 3 FTIR spectra of pure TA, AEC, pre-annealed and post-annealed AuNPs showing the involvement of various functional groups for the synthesis and stabilization of AuNPs. Figure 4 HRTEM images of AgNPs obtained from different HAuCl4.xH2O concentration (0.4 mM to 3.2 mM) at different magnifications, their corresponding SAED pattern and histogram showing the crystalline nature of AuNPs and size distribution respectively. Figure 5 Graphical representation of change in average size and shapes of AuNPs with the increase in HAuCl4.xH2O concentration from 0.4 mM to 3.2 mM, (B) linear relationship between the average size and corresponding HAuCl4.xH2O concentration. Figure 6 XPS spectra of AuNPs (A) wide scan spectra, (B) Au 4f spectrum, (C) C 1s spectrum (D) O 1s spectrum. Figure 7 Peroxidase-like activity of AuNP at pH 4 and temp 37 oC at (A) different time interval using 50 µL of 1 mM TMB, 50 µL of 1 mM H2O2 and 50 µL of AuNPs in 200 µL of NaAc buffer with inset photographs of corresponding change in color, (B) relationship between absorbance and incubation time with inset calibration plot showing the linear relationship between 2-14 min with R2=0.975, (C) different concentrations of TMB at pH 4 and temp 40 oC using 50 µL (1 mM) H2O2 and 50 µL of AuNPs in 200 µL of NaAc buffer at 18 min of fixed incubation time with inset photographs of corresponding change in color, (D) relationship between absorbance and TMB concentrations with inset calibration plot showing the linear relationship between 0.1-0.8 mM with R2=0.993. Figure 8 Peroxidase-like activity of AuNPs at pH 4 and temp 40 oC at (A) different amount of AuNPs using 50 µL (1 mM) H2O2 at constant 18 min of incubation time and 50 µL of 0.8


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mM TMB with inset photographs of corresponding change in color, (B) relationship between absorbance and AuNPs amount with inset calibration plot showing the linear relationship between 10 µL to 60 µL with R2 = 0.976, (C) different concentrations of H2O2 at constant 18 min of incubation time, 50 µL of 0.8 mM TMB and 80 µL of AuNPs with inset photographs of corresponding change in color, (D) relationship between absorbance and AuNPs amount with inset calibration plot showing the linear relationship between 2-8 mM with R2=0.997. Figure 9 (A) UV-visible spectra of AuNPs+TMB+H2O2 system showing sensitive detection of GSH in the absence and presence of its different concentrations with inset photographs showing the corresponding change in color from dark blue to almost transparent with the increasing GSH concentrations, (B) relationship between absorbance and GSH concentrations showing the linear relationship between 1 - 40 µM with R2 = 0.998, (C) detection of GSH in serum samples with inset photographs showing the respective color change and (D) their corresponding bar diagram. Table Captions Table 1 Comparison of different AuNPs based sensors for the detection of GSH. Table 2 Utilization of the developed sensor system for the detection of GSH in real sample.



Schemes Scheme 1


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Scheme 2

OH O O O

OH OH OH

O

O O O

O O

OH

OH

OH

OH

OH OH

OH OH

OH

O O

O

OH

O

O

OH

OH

OH

OH

OH OH

O OH OH

O

OH O

O

O O O

OH

OH

Tannin

O

O

OH O

O

OH

O-Au3+ O-Au3+

OH

OH

Au3+

OH

O O O

OH OH

OH OH

Au0 n

OH

n

Au

3+

Au

3+

II-Reduction Au0

I-Photoactivation

Au3+

Au0

Au0

Au0 Au0

Au0

3+

Au

0

III-Nucleation

Au3+/AEC Complex

0

3+

Au3+ Au

Gold metal ion

OH

O

n

Au3+ Au3+ Au

Au3+ Au3+ Au3+

OH O

O

OH

Au3+ Au3+ Au3+

3+ 3+ Au3+ Au Au

OH

O O

OH

OH

n

+

O O

Au0Au Au0 Au0

0 0 0 Au 0 Au0 Au Au Au0Au 0 0 Au 0Au 0 0Au Au Au Au00Au0Au 0 0Au 00 00 AuAu 0 Au 0 Au 0 AuAu 0 Au Au 0 Au 0 00 0Au 00 Au Au Au Au Au 0 0Au 0 0 0 Au Au Au Au

Au0

Au0 Au0 0

Au 0 Au0Au 00 0 0Au AuAu 0Au0 Au Au Au0

Au

Au0 0 0 Au0AuAu 0 0 0 Au Au Au 0 Au

V- Growth of AgNPs

IV-Clusture Fomation



Scheme 3


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Figure 7



Figure 8


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Figure 9



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Table 1

Probe

Mode

Linear Range

Detection limit

Detection time

References

AuNPs-ppzdtc CQDs-AuNPs SAMb-AuNPs AuNPs AEC synthesized AuNPs

Colorimetry Colorimetry Colorimetry Colorimetry Colorimetry

8-250 nM 1.0-4.0 µM 0.2-0.9 µM 0.5-1.25 µM 1-40 µM

8 nM 21.7 nM 0.5 µM 0.013 µM

30 5 min 10 min

54 6 55 56 Current Work

Table 2

Sample

Without Spiking

GSH Spiked

GSH Measured


Recovery (%)

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Serum 1 Serum 2 Serum 3 Serum 4 Serum 5

(µM) 18.84±0.94 8.50±0.42 12.85±0.64 0.69±0.03 9.66±0.48

(µM) 5 10 15 20 25


(µM) 24.25±1.21 18.00±0.90 27.30±1.36 20.35±1.02 35.35±1.77

108.2 95.0 96.3 98.3 102.8


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AuNPs synthesized form plant extract showed peroxidase-like activity for colorimetric detection of glutathione from human blood serum


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Size-Dependent Synthesis of Gold Nanoparticles and Their

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