Biosynthesis of Gold Nanoparticles by Foliar Broths: Roles of Biocompounds and Other Attributes of the Extracts
© The Author(s) 2010
Received: 11 February 2010
Accepted: 17 May 2010
Published: 15 June 2010
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© The Author(s) 2010
Received: 11 February 2010
Accepted: 17 May 2010
Published: 15 June 2010
Biosynthesis of nanoparticles has arisen as a promising alternative to conventional synthetic methodologies owing to its eco-friendly advantages, and the involved bioprotocol still needs further clarification. This research, for the first time from the standpoint of statistics, confirmed an electrostatic force or ionic bond-based interaction between the chloroauric ions and the involved bioconstituents and manifested that reducing sugars and flavonoids were both important reductants responsible for conversion of Au(III) to Au(0). The result also demonstrated that the proteins were not the reducing agents, yet they might be protection agents in biosynthesis of gold nanoparticles (GNPs). Besides, a significant linear relationship was found between the anti-oxidant ability of the foliar broths and their capability to reduce Au(III) into Au(0). Furthermore, the preliminary investigation based on the boxplot on the size/shape distribution of the biosynthesized GNPs revealed that gold nanospheres with higher degree of homogeneity in size tended to be promoted by foliar broths containing higher content of reducing sugars/flavonoids and proteins. Otherwise, i.e., for those broths with lower content of the above biocompounds, sphere GNPs of wider size distribution or even gold nanotriangles tended to be fabricated.
(See supplementary material 1)
Nanotechnology owing to its promising applications has received tremendous attention in the past decades. As building blocks in nanotechnology, various methods [1–3] have been developed to fabricate nanostructures of well-defined compositions. However, conventional physical and chemical methods either are energy intensive or impose environmental hazards due to toxic solvents or additives as well as hazardous by-products. Hence, it is of great interest to develop environmentally benign alternatives, among which biological systems arise as a typical instance. In 1999, Klaus et al.  initiated the biosynthesis of Ag nanoparticles (NPs) by Pseudomonas stutzeri AG259, and the shift from bacteria to fungus was leaded by Sastry et al. [5–7]. However, in addition to the delicate culture and storage, subsequent processing of NPs formed by intracellular biosynthesis is generally difficult, and microorganisms used for the extracellular biosynthesis of NPs must be extensively screened . In recent years, biosynthetic method employing plant extracts or biomass has appeared as a simple and viable alternative to microorganisms, e.g., plants such as coriander alfalfa , Aloe vera, Avena sativa biomass , lemongrass , Cinnamommum camphora etc. have been reported relatedly. Our group have demonstrated that a large number of plants possess the capability to convert Au(III) into Au(0) .
But there remains a significant challenge in understanding and predicting nanoparticle size and shape from a given set of biosynthetic conditions (e.g., choices of plants), which involves a full understanding of the bio-protocol. Even an accurate determination of the involved biocompounds that provides the premise for illustration of the bio-protocol could be tough. The diversity of biocompounds in the biomass makes individual purification and determination of all the biocompounds not viable. Synergic effects among these compounds might also add to the complexity. Moreover, even if for generation of the same kind of metal NPs, cases vary greatly among different bio-systems [9–13]. Consequently, a most universal explanation to account for generation of those NPs should cover as many cases as possible.
Currently, the Fourier transform infrared spectroscopy (FTIR) analyses by Huang et al.  revealed that polyols were responsible for the generation and stabilization of NPs. Among various polyols, the reducing sugars and/or the terpenoids were speculated to play a role in the bioreduction . Water-soluble heterocyclic biocompounds or proteins were considered as stabilizing ligands of the NPs [12, 13]. And the pH condition could also affect the process . There were also investigations that isolated individual biocompounds such as chitosan  and established possible mechanisms to illustrate the process.
The above studies were single organism based, focusing on individual organisms or biocompounds, and the specific information of which might not be applicable to other various cases. As well, currently the FTIR spectroscopy that mainly renders local information about related functional groups has dominated the existing methods of research, but the involved biocompounds could not be accurately determined only by FTIR since the same functional group could exist in a variety of different biocompounds. Therefore, it is imperative to explore complementary methods to illustrate the mechanism underlying biosynthesis of metal NPs.
To contribute to the determination of biocompounds involved in biosynthesis of gold nanoparticles (GNPs) by foliar broths, a statistical analysis is proposed in this work to investigate the influences of five immanent parameters of the foliar broths, i.e., the original pH value, the content of reducing sugars, flavonoids and proteins and the anti-oxidant capability, upon the Au(III) conversion and the size/shape distribution of the biosynthesized GNPs. As the parameters of the foliar broths are, respectively, evaluated, the pertinence of the research is enhanced. Moreover, due to its statistical characteristics the present research tends to be systematic. To our knowledge, this is the first report using a statistical method attempting to view bio-protocol of GNPs in a systematic perspective.
Twenty-four kinds of randomly selected plant leaves (cultivated in Fujian, China, see the supporting information) after abstersion and drying were ground into powder, respectively. In a typical preparation, a mixture of the as-prepared powder and deionized water (20 mg ml−1, 50 ml) was heated and kept boiling for 5 min. The boiled broth was allowed to cool down and then decanted. Such resulting filtrate was adjusted to 50 ml with deionized water to obtain the foliar broth for further experiments.
Chloroauric acid (HAuCl4 · 4 H2O, purchased from Sinopharm Chemical Reagent Co. Ltd, China) was used as received. During biosynthesis of GNPs, aliquot of aqueous HAuCl4 (0.04856 mol l−1) was added into the broth to obtain a final HAuCl4 concentration of 1 mM l−1. And the solution was kept in an enclosed shaker at 30°C reacting for 15 min.
The ultraviolet–visible–near infrared spectrum (UV–Vis–NIR) was conducted for characterization of GNPs. In a typical operation, an appropriate portion of the reaction mixture after dilution was transferred into a 1 × 1-cm cuvette, and the absorbance in the range of 400–1,100 nm was recorded against deionized water by the UV–Vis–NIR spectrophotometer (TU 1900/Cary 5000) with scanning step of 1 nm.
where m (ppm) denotes the residual concentration, C (mol ml−1) the initial concentration of [AuCl4]− and the coefficient 197 (g mol−1) the relative atomic weight of Au.
Original pH value of each broth was assayed with a pH meter (Delta-320, Mettler Toledo).
Spectrophotometric method was used to assay the flavonoids content in each broth . Rutin of 10 mg (dried at 105°C, purchased from Sinopharm Chemical Reagent Co. Ltd, China) was dissolved in 5 ml ethanol (95% (v/v)) in a 50-ml volumetric flask, and then the solution was diluted to 50 ml using deionized water for linear assay to establish the calibration line.
In a typical determination, firstly, a combination of aqueous NaNO2 (0.4 ml, 5 wt%) and 1 ml adjusted sample solution (depending on the approximate content of the flavonoids in each broth, concentrations of the broths herein used were already adjusted accordingly with deionized water such that the final sample could be within the linear range of the assay, likewise for the case of reducing sugars and proteins) was agitated in a volumetric flask of 10 ml. Then, the solution was left stand for 6 min to allow for sufficient interaction between the added reagents and the biocompounds (which was also the reason for the same treatment hereinafter). Afterward, aqueous Al(NO3)3 (0.4 ml, 10 wt%) was pipetted into the mixture. Such resulting solution after agitation was kept stationary for 6 min, and subsequently NaOH solution (4 ml, 4 wt%) was transferred into it. After being diluted to 10 ml and agitation, the final solution was allowed to stand for 15 min. Finally, its absorbance at 510 nm was recorded using Visible Spectrophotometer (DU7400, Beckman Coulter, Inc.) with mixtures of above additives served as blank.
The DNS (3, 5-dinitrosalicylic acid) method was employed to determine the reducing sugars content in each broth. A combination of 1.0 ml modified broth and 2.0 ml DNS reagent was bathed in boiling water for 10 min and then was cool down using flowing water. After addition of 10 ml deionized water, the absorbance of the final solution at 540 nm was measured against DNS reagent/water blank using Visible Spectrophotometer (DU7400, Beckman Coulter, Inc.). Aqueous glucose was used as standard solution to obtain a calibration line.
Coomassie brilliant blue method was used for measurement of the proteins content in each broth. A portion (5.0 ml) of Coomassie brilliant blue G-250 dye reagent (0.01% (W/V)) was added into 1.0 ml modified broth. The mixture was agitated and kept stationary for 2 min. The absorbance of the final sample at 595 nm was measured against the dye reagent/water blank by Visible Spectrophotometer (DU7400, Beckman Coulter, Inc.). Bovine serum albumin (BSA, BR, Livzon Pharmaceutical Group Inc.) as standard solution was employed to establish the calibration line.
where A 0 A 1 and A 2 are absorbance of the blank, the sample and the control, respectively.
The SR50 value, which denotes the concentration of the leaves required to remove 50% DPPH radicals in the solution, was calculated by linear regression of plots where the abscissa represented the concentration of the leaves and the ordinate the DPPH radical scavenging rate.
Triplicates were conducted in each assay of the parameters.
From the critical value of correlation coefficient ρ = 0 table  the two critical values, i.e., r 0.05,22 and r 0.01,22, were found to be 0.404, 0.515, respectively.
In addition, for the primary investigation into the size/shape distribution of biosynthesized GNPs, the boxplot was used with five-number summaries, i.e., the smallest observation, the lower quartile and the upper quartile cutting off the lowest and highest 25% of the data, respectively, the median which is the middle value of the data and the sample maximum . The boxplot is based on robust statistics which are more resistant to the presence of outliers than the classical statistics based on the normal distribution . Hence, the data sets of the parameters could be described without any statistical assumption and the difference between data sets, if there are any, could be reflected directly.
During biosynthesis of GNPs, all of the ultimate reaction solutions possessed the characteristic red color, indicating generation of GNPs which was also validated by the UV–Vis–NIR characterizations (see supporting information). Such resulting GNPs were built upon Au(III) conversion, a redox reaction depending on the properties of the broths (as the reaction time, temperature and pressure were fixed). Accordingly, the relevancies between the conversion of Au(III) and each parameter, e.g., original pH value, the content of flavonoids, reducing sugars and proteins, as well as the anti-oxidant capability of the foliar broths should reflect the role of each parameter upon the biosynthesis of GNPs, as demonstrated in the following sections.
For the research where pH value was the center of attention , with choice of biomass and other conditions fixed, the pH value of the solution was controlled to be the predominating factor influencing the interaction between [AuCl4]− and the biomass. However, herein multiple other factors varying both in quantity and quality among individual plants might regulate the conversion in a pattern much stronger than that of the original pH values. Furthermore, the pH value in the former research was modified by the inorganic acid/alkali to extend from relatively strong acidic to weak or even to alkaline conditions. Nevertheless, the so-called original pH was the active acidity denoting the concentration of dissociated natural organic acids, and most of the foliar broths were weakly acidic with pH from ca. 4.1 to 7.6. Therefore, the effect of the original pH conditions on Au(III) conversion was not evident within the range.
Accordingly, such a linear relationship of relative significance in the statistical perspective verifies flavonoids as, or among, the biocompounds responsible for reducing Au(III) into Au(0), supplementing the none-typical information regarding the flavonoids from the FTIR analysis . Besides, without exception, in this study foliar broths with relatively denser flavonoids presented higher Au(III) conversion, e.g., when flavonoids content was above 1.25 mg ml−1, the responding conversions were over 95%. Therefore, content of flavonoids of the plants, since which has already been both extensively and intensively investigated , could be an index for preliminary evaluations of the untapped plants in terms of biosynthesis of GNPs.
There were already precedents using purified reducing sugars to reduce metallic ions, which circumvented the complicacy encountered by those using foliar broths. For instance, Ag+ was reduced by glucose in the nanoscopic starch template , and the fructose was demonstrated to be the best-suited reducing agent over other sugars [27, 28]. These results involved with isolated reducing sugars on one hand supported the statistical result here introduced on the other hand guaranteed interaction between the two. For instances, information from the former such as the binding pattern , the stabilization [27, 28] regarding the biocompounds and the NPs might also be available to the present one where alike biocompounds interact and bind with the metal NPs.
Compared with the polyols, the case of the proteins seemed to be more complicated. For instance, when camphora leaves were used in fabrication of Au or Ag NPs, the proteins seemed to exhibit little importance , neither they did in the case using neem leaves . However, Ag NPs were synthesized and stabilized successfully by cyclic peptides in latex of Jatropha curcas. And biomimetic synthesis and patterning of Ag NPs using targeted peptides  was also conducted. In both cases, the peptides were believed to function as both the reducing and protection agents.
It has been found during the experiments that the proteins content in the foliar broths is relatively low, which on average is only one-twelfth and one-seventh of that of the reducing sugars and the flavonoids, respectively. And yet the quantity of amino acid residues such as cysteine , which are believed to interact with or to reduce Au(III) into Au(0), is even less. As a consequence, in the redox reaction the polyols as well-established reductants would serve as the principal electron donor, leading to the poor linear correlation between the proteins content and the conversion of Au(III) . Hence, the present result does not necessarily contradict against aforementioned researches involving peptides as reducing agents [29, 30]. As well, since the reduction of Au(III) and the stabilization of the GNPs are two distinguished aspects of the process, the result neither invalidate proteins as capping agents to prevent the GNPs from aggregation in the green protocol.
In summary, being parameter targeted, the methodology demonstrated strengthens the pertinence with respect to biocompounds involved in biosynthesis of GNPs. The linear relationships with different levels of significance between the conversion of Au(III) and immanent parameters of the broths not only contributed to determination of biocompounds involved in biosynthesis of GNPs, but also revealed the similarities among numerous individual plants in terms of biosynthesis of GNPs, which implied the existence of an uniform mechanism underlying this universally spontaneous phenomenon.
Generally, the UV–Vis–NIR spectrum patterns could be sorted into two categories through statistical grouping. For group 1, each of the absorption patterns presented only one well-defined and shape-constant absorption band with maximum absorbance located at 500–600 nm. And these relatively narrow bands were associated with sphere GNPs with high degree of homogeneity in size . For group 2, besides the absorption band at 500–600 nm, the spectrums either have, or show the tendency to have, another band in 600–1,100 nm, suggesting generation of sphere GNPs with wide size distribution or particle aggregation or even existence of gold nanotriangles [34, 35] (see the supporting information).
That is, in the biosynthesis of GNPs by foliar broths sphere GNPs with higher size homogeneity were promoted by higher average reducing rate, while the lower one leaded to GNPs with wider size distribution or even gold nanotriangles. Explanations of such phenomenon involve with the nucleation and crystal growth stages during synthesis of GNPs. From the stand point of kinetics, in group 1 the reduction of Au(III) to Au(0) due to higher content of reducing agents was faster than that in group 2. This leaded to denser nucleation which therefore predominated over the growth of the GNPs and as a result prevented the gold atoms and clusters formed at early stages of the reaction from growing into extremely large particles .
However, fast nucleation could not work solely to generate uniform GNPs spheres considering their high instability due to high surface Gibbs energy. Denser substances for passivation to prevent GNPs from aggregation were expected in group 1 than the other. Higher concentration of the reducing agents and/or their responding products resulted from reduction of Au(III) might be an important resource of the protection agent  contributing to the higher size homogeneity of GNPs in group 1. What is more, the proteins concentration that in the former section was observed with little importance as reducing agents, however, herein in general higher in group 1 than group 2. This suggests that the proteins might also be the protection agents due to their strong affinity to bind metals possessed by carbonyl groups from the amino acid residues and peptides of proteins .
Additionally, it could be seen that the wavelength of the maximum absorbance in the UV–Vis–NIR spectrums varied from plant to plant, indicating that spherical GNPs of various sizes and triangular GNPs might be obtained. Thereby through adjusting the choice of the plants, biosynthesis of spherical or triangular GNPs might be size controllable, which could be of great environmental and operational advantages over those chemical methods employing additives for adjustment .
In summary, this statistical investigation supported the speculation that the [AuCl4]− interacted with the biocompounds through an ionic bond or an electrostatic force, and both reducing sugars and flavonoids were proved to be important reductants responsible for the conversion of Au(III). The research also excluded the possibility for the proteins to be reductants yet it indirectly supported them as the protection agent in the biosynthesis of GNPs by foliar broths. As well, a significant linear relationship between the anti-oxidant activity of the foliar broths and their capability to reduce Au(III) into Au(0) was discovered. Besides, the preliminary analysis regarding the size/shape distribution of the biosynthesized GNPs revealed that the foliar broth containing higher content of reducing sugars/flavonoids and proteins in general supported formation of sphere GNPs with higher homogeneity in size while otherwise sphere GNPs with wider size distribution or even nanotriangles might be developed. Not only this statistical analysis could complement the conventional optical spectrum methodologies to investigate biocompounds involved in biosynthesis of GNPs, but also it could contribute to exploration of alternatives in rough screening of the affluent plant resources in terms of fabrication of GNPs.
This work was supported by the National High Technology Research and Development Program of China (863 Program, Grant No. 2007AA03Z347), the National Natural Science Foundation of China (Grant Nos. 20576109, 20776120 and 20976146) and the Natural Science Foundation of Fujian Province of China (Grant No. 2008J0169).
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