Green-assisted tool for nanogold synthesis based on alginate as a biological macromolecule

Abstract

Large-scale biomedical applications of nanogold reflect the challenge faced by recent researches in the investigation of green synthesis methodologies, which are mostly complicated and/or expensive processes. The present report offers a totally green method using a quite simple and costless technique for the manufacture of Au nanoparticles based on alginate macromolecules. Hydrogen peroxide was used for the oxidative degradation of alginate at room temperature to produce more accessible fragments, beneficial for the reduction of Au ions to Au⁰ and as a stabilizer for the produced nanogold. The competence of the mentioned procedure was tested by comparison with the alkali/glucose/alginate system, in which glucose and alginate were used as a reducer and stabilizer, respectively. For both the systems, surface plasmon resonance peaks for nanogold were detected and similar absorbance spectra were observed. Using either 30 mmol L⁻¹ H₂O₂ or 1 g L⁻¹ glucose, similar reducing sugar content (0.32 g L⁻¹) resulted from 1 g L⁻¹ alginate. Nanogold manufactured by H₂O₂ exhibited smaller size (3.7 ± 1.1) with narrower size distribution (1.5–8.0 nm) than that produced in the case of glucose. However, enlarged nanogold was observed after storage for 5 months, which still maintained the nano dimension. The produced nanogold via the completely green technique using H₂O₂/alginate could be safely used in biomedical applications.

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1. Introduction

Gold nanoparticles (AuNPs) have multidisciplinary applications due to their unique catalytic,¹ optical and physical characteristics.^2–10 It was found that composites of nanogold have reemerged as leading candidates in the field of bio-nanotechnology, as they can be easily manufactured with a desirable particle size from 0.8 to 200 nm. Due to their ease of preparation, they can be modified to impart various functionalities, good biocompatibility and can be readily conjugated to proteins and other molecular species without altering the biological activity of the conjugated species.¹¹ Additionally, AuNPs have unique optical properties such as distinctive extinction bands in the visible region, due to the surface plasmon oscillation of free electrons.¹² These mentioned properties allow the use of AuNPs for many applications, e.g., they can be used as Raman sensors,¹³ photo-catalysts¹⁴ and photo-electrochemical materials.^15–18 For medical purposes, AuNPs are used as immune-staining markers for electron microscopic images, and as chromophores for immunoreactions and nucleic acid hybridization.^19,20 Recently researchers have studied the application of AuNPs for gene delivery into cells.^21–24 Also, AuNPs have been greatly considered as photo thermal agents in hyperthermia.²⁵

Due to the unique properties and large-scale applicability of AuNPs, several synthetic approaches have been carried out, such as chemical reduction, UV photo-activation, laser induction, lysozyme-directed generation, antibiotic mediated synthesis, bio-reduction, etc.^26–32 However, the development of simple techniques for the synthesis of AuNPs with desirable particle sizes, shapes and functions is a field of interest nowadays, and an intellectually rewarding task.^32–34 Nonetheless, the nanoparticles' stability and good dispersibility for preventing aggregation are still important issues that must be carefully manipulated. Mostly, the techniques used could be described as expensive and multi-step methodologies with undesirable ecological impacts. In some cases, they mainly depend on using hazardous chemicals/solvents, and also are not economical because of the use of expensive chemicals and/or large energy consumption. Moreover, some of these procedures generally failed in the production of small and size controlled AuNPs.

The manufacture of metal nanoparticles based on biopolymers has been widely studied,^26–49 and hydrophilic composites of natural polymers and metal nanoparticles (MNPs) have been investigated and found to have long circulation times in the human blood stream.^50,51 Production of size controlled AuNPs, with high stability and good dispersibility in colloidal form could be preceded by using commercial and environmentally beneficial biopolymers.^52–55 Polysaccharides are well known as renewable, biodegradable and biocompatible organic masses with wide applicability in the biomedical field. Alginate biopolymer, as an example of polysaccharides,^70,71 is commonly employed for multiple biomedical applications, such as cell culture, tissue engineering, drug release, immobilization of enzymes or metals for catalysts.^56,57 Alginate hydrogels are cost effective, mild reducers, biodegradable and biocompatible colloids, which have been designed to be excellent stabilizers and templates for metal nanoparticles synthesis, such as Ag, Co, Ni and iron oxide.^46,58,59 Although sodium alginate hydrogels are quite suitable as a scaffold matrix for metal nanoparticle manufacturing, only a few studies on the application of alginate in the reduction and stabilization of AuNPs have been reported.⁶⁰

Herein, a totally green method is presented for the synthesis of AuNPs using H₂O₂/alginate biopolymer in neutral medium and at room temperature. The represented method is characterized as a facile, one-pot, cost effective and energy saving approach to AuNPs synthesis. After oxidation with H₂O₂, oxidized alginate is concurrently used as a reductant for Au ions and a stabilizer for the so-obtained AuNPs. A comparative study on the preparation of AuNPs by using an alkali/glucose/alginate system was introduced as an approved experimental plan for determining the efficiency of the H₂O₂/alginate system. Reducing sugar content and UV-visible spectra for reaction mixtures prepared under different experimental conditions are systematically presented and studied. TEM images and zetasizer data for the manufactured nanogold colloids are shown and discussed. Size distribution and poly-dispersity index are both evaluated. Further confirmation of the synthesis of AuNPs was carried out using the X-ray diffraction (XRD) and Fourier Transform infrared (FTIR).

2. Experimental

Chemicals and materials

Gold chloride (AuCl₃, El-Gomhoria Pharmaceutical Chemical Company, Cairo-Egypt), sodium alginate (91–106%, Loba Chemie, Mumbai, India), sodium hydroxide (NaOH) and glucose (C₆H₁₂O₆) (Merck, Darmstadt, Germany), 3,5-dinitro-salicylic acid (C₇H₄N₂O₇) and phenol (C₆H₆O) (El-Nasr Pharmaceutical Chemical Company, Cairo, Egypt), sodium sulphite (Na₂SO₃, S.D. fine-CHEM Ltd, Mumbai, India), potassium sodium tartrate (KNaC₄H₄O₆·4H₂O, Mallinckrodt. Inc., Paris, France), and hydrogen peroxide (H₂O₂, 30%, Egyptian Company of Chemicals and Pharmaceuticals, 10^th of Ramadan, Egypt) were all used without further purification.

Manufacturing of AuNPs using alginate

Two different systems were used to prepare AuNPs based on alginate biopolymer. The main system used was H₂O₂/alginate as a totally green system, and the alkali/glucose/alginate system was used for comparing and evaluating the efficiency of the H₂O₂/alginate system.

Alkali/glucose/alginate system. Set amounts of sodium alginate (0.2–6.0 g L⁻¹) were solubilized in distilled water using a magnetic stirrer, and glucose (0.2–6.0 g L⁻¹) was then added. After complete dissolution, 100 mmol L⁻¹ of AuCl₃ were added drop wise with stirring and then NaOH (25 mmol L⁻¹) was added to the mixture. In all experiments, the total volume of reactants was fixed at 50 mL. The reaction was conducted at room temperature (≈30 °C) and 70 ± 3 °C under continuous stirring for 30 minutes. After addition of NaOH, the reaction medium acquired a reddish to bluish color, which became more intense with time, signalling the nucleation of Au nanoparticles.

H₂O₂/alginate system. Using a magnetic stirrer, definite amounts of sodium alginate (0.2–3.0 g L⁻¹) were solubilized in distilled water and then H₂O₂ (10–60 mmol L⁻¹) was added. Finally, 100 mmol L⁻¹ of AuCl₃ were added dropwise (keeping in mind that the total volume of reactants is 50 mL). The reaction was kept under continuous stirring for 30 minutes at room temperature (≈30 °C). After addition of the AuCl₃ solution, the reaction medium acquired a reddish to bluish color and darkened with time, indicating the formation of Au nanoparticles.

In both systems, the progression of the reaction was controlled by UV-visible absorption; aliquots from the bulk reaction were withdrawn at given time intervals and the absorbance spectra were evaluated.

Reducing sugar content

Alginate was degraded by the action of alkali catalysis or H₂O₂ oxidative degradation to give smaller fragments containing sugar moieties. The residual reducing sugar content produced in the mixture was detected photometrically. During the redox reaction between the Au³⁺ and alginate fragments, the aldehydic and/or ketonic groups of the fragments were fully oxidized to carboxylic groups. In the reducing sugars test, 3,5-di-nitro-salicylic acid was reduced to 3,5-di-amino-salicylic acid units. The test was carried out according to Sumner, as follows: in capped test tubes, 3 mL of DNS reagent were added to 3 mL of gold nano-colloidal solutions manufactured under different experimental conditions. The mixture was heated at 90 °C for 15 minutes. One mL of Rochelle salt was added for stabilizing the resulting color. After cooling, the absorbance was detected photometrically at 575 nm.⁶¹ Glucose was used instead of alginate solution to prepare calibration solutions in the range of 0–5 g L⁻¹. For the calibration, R² was 0.98 and the slope was evaluated to be 0.59.

Characterization of gold nanoparticles

UV-visible spectra. Nanogold colloids manifested an intense absorption peak due to the surface plasmon resonance (SPR). Hence, the UV-visible absorption spectra were used for ratifying the production of AuNPs in colloidal form under different experimental conditions. The UV-visible absorption spectra of AuNPs colloidal solutions were spectrophotometrically recorded in the wavelength range of 250–750 nm, using a multi channel spectrophotometer (T80 UV/VIS, d = 10 mm, PG Instruments Ltd, Japan).

Transmission electron microscopy (TEM). The topographical features and size distribution of the so-produced AuNPs were viewed using a JEOL-JEM-1200 high resolution transmission electron microscope from Japan, with an electron beam from Oxford instruments. Nanogold colloids were carefully added on a 400 copper grid coated by carbon film, and then evaporated in atmospheric air before conducting microscopic analysis. The mean size and size distribution of AuNPs were calculated by 4pi analysis software, using TEM photos. The average particle size was determined from the diameter of at least 50 particles.

Zetasizer. The average size, size distribution and poly-dispersity index of AuNPs were all evaluated using a Zeta sizer device (Malvern Zetasizer Nano ZS, from Malvern Instruments Ltd–UK). The instrument was attached to a He–Ne laser lamp (0.4 mW) at a wavelength of 633 nm. Measurements were performed at 25 °C in an insulated chamber using the dynamic light scattering technique.

Sedimentation of AuNPs. The prepared nanogold was precipitated from a colloidal solution in the form of the AuNPs–alginate composite. The sedimentation process of the composite was carried out by the addition of absolute methanol to the colloidal solution and then the precipitate was isolated by filtration. The precipitate was dried in an oven at 70 °C before any further analyses.

X-ray diffraction (XRD). Native alginate and the deposit of the AuNPs–alginate composite were both fixed in an X-ray diffractometer from Philips (X'Pert MPP with a type PW 3050/10 goniometer). Diffraction patterns were measured in the diffraction angle (2θ) range of 10–80°, using a PC computer with the programs PROFIT and mono-chromatized (Mo Kα X-radiation at 40 kV, 50 mA and λ = 0.70930 Å) with a step size of 0.03° at room temperature.

Fourier transformation infrared (FTIR). Infrared spectra were used to characterize native alginate and the deposit of the AuNPs-alginate by using an infrared spectrometer (Jasco FT/IR 6100) attached to a deuterium-tri-glycine sulfate (TGS) detector. The spectra were collected in the wave number range of 4000–350 cm⁻¹ using transmission mode (T%), resolution of 4 cm⁻¹ with 2 mm s⁻¹ scanning speed and 1 cm⁻¹ interval scanning.

3. Results and discussion

Regardless of the cost effectiveness, most researchers are looking for green methodologies in principle, for the preservation of human life. In the field of nanotechnology, the main challenge is the investigation of green manufacturing procedures without any undesirable byproducts. Recently, numerous reported studies concerned with the synthesis of metal nanoparticles have described their designed manufacturing processes as green techniques; however, these processes suffer from some disadvantages.^{37–39,42–46} The use of high alkalinity and elevated temperature, cause the mentioned methods not to be green enough, and their applications are accompanied by some limitations, especially in biomedical applications.

The present study is a totally green method for the synthesis of AuNPs using the H₂O₂/alginate system in neutral medium and at room temperature. Alginate as a natural biopolymer is characterized as a biodegradable and biocompatible polymer. H₂O₂ is known as a powerful oxidizing agent, which is simply degraded at room temperature, producing the safe species, O₂ and water. The efficiency of the H₂O₂/alginate system in the nanogold preparation was tested by comparing with the alkali/glucose/alginate system.

Reaction mechanism

The reaction mechanisms for the preparation of AuNPs using two designed systems are proposed and presented in Fig. 1, and could be illustrated as follows:

Fig. 1 Schematic representing the preparation mechanism of AuNPs using (a) NaOH/glucose/alginate and (b) H₂O₂/alginate.

Alkali/glucose/alginate system. In this designed reaction system, glucose, one of the most powerful natural reducers, was used to reduce Au³⁺ ions to generate AuNPs. However, sodium alginate, a linear copolymer of 1,4-linked β-D-mannuronate (M) and α-D-guluronate (G) residues, was used as a stabilizer for the net produced AuNPs. Fig. 1a shows the mechanism of the preparation of the AuNPs, using an alkali treated glucose/alginate system that involves four main steps as follows: (i) an ion exchange interaction between Au³⁺ and terminal carboxylic groups of alginate was preceded by mixing Au³⁺ salt with certain amounts of the glucose/alginate mixture. (ii) After alkali addition, the alginate macromolecules were degraded and de-polymerized, giving the original monomers as fragmented units. Catalytic degradation of alginate was activated by the action of Au³⁺ ions. (iii) Glucose units reduced Au³⁺ ions to Au⁰ in the nano dimension and the redox reaction was motivated in alkaline medium. Additionally, AuNPs nucleation was induced by alkali action.³⁹ (iv) Due to the high polarity of water, AuNPs usually agglomerate immediately and form enlarged particles; however, alginate fragmented units stabilize the manufactured AuNPs from further agglomeration. Stabilization might be carried out in two ways, firstly, by coordination of Au⁰ by COO⁻ and O⁻ groups, then, the fragmented macromolecules result in a colloidal solution crowded with reducing fragments, which might protect the manufactured NPs from coagulation, through their steric effects.

H₂O₂/alginate system. In this designed system, hydrogen peroxide was added to the alginate in order to enhance its reducibility and accessibility, to play the dual role of reducer of Au³⁺ ions and stabilizer for AuNPs. The significant characteristic of H₂O₂ is its decomposition at room temperature to oxygen gas and water,⁶² which are safe and harmless species. Fig. 1b shows the alginate oxidation reaction, which involved four main steps as follows: (i) H₂O₂ was decomposed to oxygen gas and water. (ii) Sodium alginate was degraded by the action of oxygen gas to produce fragmented units (oligomers) lowered in molecular weight. Oxidative degradation of alginate was further catalyzed by the action of Au³⁺ ions. (iii) Ion exchange interactions between Au³⁺ and the terminal carboxylic groups of alginate fragments take a place with the drop wise addition of Au³⁺ salt solution to the oxidized alginate.⁴⁵ (iv) The produced fragments are higher in accessibility/reducibility and hence, fully reduced Au³⁺ to Au⁰. Alginate fragmented units concurrently protect the generated AuNPs from further agglomeration, through coordination and steric effect. Their powerful chelating sites (COO⁻ and O⁻) may also aid in protecting NPs via coordination. While the produced fragmented macromolecules may protect AuNPs from aggregation through their steric effects, which in turn resulted in the bio-mixture wrapped gold. Additionally, using H₂O₂ causes the pH value of the reaction medium to be close to 8. This pH value is supposed to slacken the catalytic degradation of alginate, giving limited amounts of reducing units and subsequently, slowing down the redox reaction between alginate reducing fragments and Au ions. Hence, the nucleation of AuNPs was carried out slowly, resulting in the production of well-dispersed and size controlled AuNPs.

Reducing sugars contents

Monitoring of the reducing sugar content in the reaction medium is a very important issue for approving the suggested reaction mechanism for manufacturing AuNPs. During the redox reaction between sugars (either glucose or fragmented products of alginate) and Au³⁺, the Au³⁺ ions were reduced to Au⁰, and the sugars with alcoholic functional groups were oxidized to the first oxidized form with more aldehydic and/or ketonic groups. Hence, in the reducing sugars test, the first oxidized form of reducing sugars in the reaction media were fully oxidized to carboxylate species, and 3,5-dinitrosalicylic acid was reduced to 3,5-diaminosalicylic acid, which was photometrically detected. The increment in the reducing sugar content means that the redox reaction between alginate and Au³⁺ ions is in progress, which in turn reflects the increment in the affinity of AuNPs production. For blank solutions (in the absence of Au³⁺), nearly no reducing sugars were produced, which confirmed the main role of Au³⁺ ions in catalyzing the degradation of alginate in the presence of alkali or hydrogen peroxide.

Table 1 shows the effect of using the two designed systems under different experimental conditions on the concentration of the produced reducing sugars. In the case of the alkali treated glucose/alginate system, an increment in the reducing sugar content was observed on raising the concentration of alginate from 0.2 g L⁻¹ to 2 g L⁻¹. Prolonging the reaction duration to 30 minutes resulted in higher reducing sugar content (it reached 0.45 g L⁻¹). Production of reducing sugars was further increased with both concentrations of glucose and alginate. Increasing the glucose/alginate mass ratios from 0.2 : 0.2 g L⁻¹ to 1 : 1 g L⁻¹ was accompanied by a boost in reducing sugar content. With a further increase in mass ratio to 2 : 2 g L⁻¹, a marginal decrease in reducing sugar content was observed, which could be attributed to polymer destruction.⁶³ By raising the reaction temperature from room temperature to 70 °C, the reducing sugar content was significantly increased, as thermal energy is logically thought to increase the rate of alginate alkali hydrolysis and liberate more reducing fragments.

Table 1 Reducing sugar content as a function of the activated system in the presence of 100 mmol L⁻¹ of AuCl₃^a

System	NaOH conc. (mmol L^−1)	Glucose conc. (g L^−1)	H2O2 (mmol L^−1)	Temp. (°C)	Alginate (g L^−1)	Reducing sugar (g L^−1)
						1 min	30 min
a For blank samples: in the absence of AuCl3, the reducing sugar content ≤0.01 g L^−1, for blank samples: for glucose alone with alkali, reducing sugar content = 0.11 g L^−1.
Glucose/NaOH	25	0.2	—	RT	0.2	0.08 ± 0.03	0.28 ± 0.02
	25	0.2	—	RT	1.0	0.22 ± 0.03	0.49 ± 0.02
	25	0.2	—	RT	2.0	0.40 ± 0.02	0.50 ± 0.03
	25	1.0	—	RT	1.0	0.27 ± 0.02	0.32 ± 0.02
	25	1.0	—	70	1.0	0.42 ± 0.02	0.28 ± 0.02
	25	2.0	—	RT	2.0	0.54 ± 0.02	0.21 ± 0.02
	25	1.0	—	RT	2.0	0.48 ± 0.02	0.29 ± 0.02
H2O2	—	—	10	RT	0.2	0.21 ± 0.01	0.28 ± 0.03
	—	—	10	RT	1.0	0.19 ± 0.01	0.22 ± 0.02
	—	—	30	RT	1.0	0.37 ± 0.02	0.32 ± 0.02
	—	—	30	RT	3.0	0.30 ± 0.03	0.35 ± 0.03

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In the case of the H₂O₂/alginate system, it was observed that there was no significant difference in reducing sugar content between 1 and 30 minutes, reflecting the rapid degradation of alginate. This is related to the powerful effect of H₂O₂ in the oxidative degradation of alginate, which appeared from the first minute. This phenomenon explains the high reducing sugars obtained after 1 minute at low alginate concentration, compared to that at using alkali/glucose. On the contrary, with using alkali/glucose and H₂O₂, the reducing sugar content was not significantly increased with increased alginate content. However, the reducing sugar content was considerably increased from 0.19 g L⁻¹ to 0.37 g L⁻¹ by increasing the concentration of H₂O₂ from 10 mmol L⁻¹ to 30 mmol L⁻¹, which could be attributed to the insufficient amounts of hydrogen peroxide used. This further confirmed the powerful oxidation effect of hydrogen peroxide in alginate degradation and hence, concentrated H₂O₂ led to the liberation of greater amounts of sugar species. Generally, the reducing sugar content was much greater in the case of using the alkali/glucose/alginate system than the H₂O₂/alginate one. This may attributed to the fact that in the former, glucose being one of the reactors already has an effect on the net result of the reducing sugar content, by raising it.

Using both of the designed systems exhibited a conceptualization of the role of alkali and hydrogen peroxide in manufacturing nanogold colloids using alginate biopolymer. NaOH, a strong alkali, degraded alginate immediately after addition. However, H₂O₂ as a strong oxidizing agent exhibited a much faster rate of alginate degradation. Using H₂O₂ to degrade alginate is much preferred because of the liberation of safe/harmless oxidizing species (O₂ and H₂O). Characterization of the synthesized AuNPs colloidal solutions was spectrophotometrically and microscopically carried out and the data are presented and discussed in the following sections.

UV-vis spectroscopic analysis

UV-visible spectra were detected in the absorbance range of 250–750 nm. Regardless of the parameters for synthesis of both designed systems, UV-visible analyses have shown that Au/alginate solutions exhibited a surface plasmon absorption band, peaking in the wavelength range of 510–540 nm (Fig. 2 and 3). These results correspond to a plasmon resonance effect originating from the quantum size of AuNPs and thus confirmed the synthesis and nucleation of Au nanoclusters in both designed system solutions.^32,35 The peak position and peak intensity were both dependent on the experimental conditions of the redox reaction between Au ions and reactors.

Fig. 2 Absorbance spectra of the AuNPs colloidal solution using 100 mmol L⁻¹ AuCl₃ (a) at different times, using 2 g L⁻¹ alginate, 0.2 g L⁻¹ glucose and 25 mmol L⁻¹ NaOH at room temperature; (b) at different concentrations of alginate, 0.2 g L⁻¹ glucose and 25 mmol L⁻¹ NaOH at room temperature after 30 minutes; (c) at different molar ratios of glucose/alginate, using 25 mmol L⁻¹ NaOH at room temperature after 30 minutes; (d) at different temperatures using 1 g L⁻¹ alginate, 1 g L⁻¹ glucose and 25 mmol L⁻¹ NaOH after 30 minutes.

Fig. 3 Absorbance spectra of the AuNPs colloidal solution, using 100 mmol L⁻¹ AuCl₃ (a) at different times, using 1 g L⁻¹ alginate and 30 mmol L⁻¹ H₂O₂ at room temperature; (b) at different alginate concentrations, using 30 mmol L⁻¹ H₂O₂ at room temperature after 30 minutes; (c) at different H₂O₂ concentrations, using 1 g L⁻¹ of alginate at room temperature after 30 minutes.

For the alkali/glucose/alginate system, increasing the reaction time to 30 minutes, exhibited non-notable changes in the absorbance values (Fig. 2a). Intensity and sharpness of the AuNPs absorbance band was increased with alginate concentration from 0.2 g L⁻¹ to 2.0 g L⁻¹, but further increment in the alginate concentration to 6.0 g L⁻¹ led to peak broadening. This is related to the amount of AuNPs, which increased by increasing the alginate concentration; this foundation confirmed the effect of alginate in the complete reduction of Au ions. However, using concentrated alginate (>2 g L⁻¹) might be reflected in enlarged capped clusters, which are supposed to coagulate and form larger sized AuNPs, resulting in more peak broadening. Increasing the glucose/alginate mass ratios from 0.2 : 0.2 g L⁻¹ to 2 : 2 g L⁻¹ resulted in a significant increment in the absorbance peak intensity and sharpness. Increasing the glucose/alginate mass ratios may be accompanied by the production of greater amounts of AuNPs with smaller size. These results are in agreement with the reducing sugar data.

Increasing the reaction temperature to 70 °C resulted in a decrement in the absorbance values and peak sharpness, which might be explained by the following two reasons. (i) Increasing the reaction temperature may result in increasing the rate of generation of reducing fragments faster than the reduction of Au ions. (ii) Elevated temperature leads to an increase in the kinetic energy of the so-produced AuNPs, which consequently increases the rate of collisions between particles that then start to collapse, tending to aggregate.

For the other designed system of the H₂O₂/alginate system, the absorption spectral data are shown in Fig. 3. The height and sharpness of the absorption peaks were not significantly changed by increasing the reaction duration from 1 to 30 minutes or increasing alginate concentration from 1 g L⁻¹ to 3 g L⁻¹. This is in agreement with the results of the reducing sugar content, which showed the powerful effect of H₂O₂ in the rapid degradation of alginate, from the first minute. The gradual increment in H₂O₂ from 10 mmol L⁻¹ to 30 mmol L⁻¹ was accompanied by an enlargement in absorbance values and peak sharpening, which is in harmony with the data recorded for reducing sugar content. This finding confirmed the role of H₂O₂ in the oxidative degradation of alginate to produce more reducible fragments, which is in turn, applicable for manufacturing AuNPs. Consequently, the amount of the so-obtained AuNPs was enhanced by increasing H₂O₂ concentrations. However, further increment in H₂O₂ concentration to 60 mmol L⁻¹ resulted in a decrease in absorbance. This might be explained by the aggressive oxidizing effect of highly concentrated peroxide, resulting in the production of alginate fragments, much smaller in molecular weight, which are unable to manufacture gold nanoparticles.

Morphology, particle distribution and poly-dispersity

The manufacture of AuNPs in a well-dispersed and stable colloidal form was further confirmed and characterized by monitoring the morphological features and size distribution using a transmission electron microscope and a zetasizer instrument. The size distribution of the AuNPs was calculated from TEM microscopic images and from zetasizer data. These measurements were carried out to investigate the efficiency of alginate to prepare nanogold, in addition, the aging effect was studied under different conditions, due to its importance in the application of the prepared AuNPs.

Fig. 4 and 5 represent TEM images and size distribution results for the obtained AuNPs using alginate through two different designed systems. From TEM micrographs, spherical AuNPs were obviously seen and were well distributed. In the case of using the alkali/glucose/alginate system, a low concentration of glucose (0.2 g L⁻¹) produced Au particles in the size range of 3–34 nm, with a mean size of 23.3 ± 13.1 nm (Fig. 4a and Table 2). However, by increasing the glucose concentration to 1 g L⁻¹, a narrower size distribution was detected (3–16 nm) with smaller average size of 9.4 ± 2.7 nm (Fig. 4b and Table 2). This reflects the main role of glucose in the reduction of Au ions, which was consequently confirmed by producing smaller sized AuNPs using a higher concentration of glucose, which is in harmony with the reducing sugar data. By storing the sample for 5 months, some agglomeration occurred, achieving enlargement in size distribution and particle size, as the mean size obtained was 29.0 ± 14.1 nm (Fig. 4c and Table 2).

Fig. 4 TEM and zetasizer images for AuNPs colloidal solution using the glucose/NaOH system, 100 mmol L⁻¹ AuCl₃ and 25 mmol L⁻¹ NaOH after 30 minutes at RT, (a) 0.2 g L⁻¹ glucose and 2 g L⁻¹ alginate; (b) 1 g L⁻¹ glucose and 1 g L⁻¹ alginate, fresh; (c) 1 g L⁻¹ glucose and 1 g L⁻¹ alginate, stored for 5 months. Size distribution is shown for the corresponding images and fitting curves are introduced as blue lines.

Fig. 5 TEM and zetasizer images for AuNPs colloidal using H₂O₂, 100 mmol L⁻¹ AuCl₃, 25 mmol L⁻¹ NaOH after 30 minute at RT, (a) 10 mmol L⁻¹ H₂O₂ and 1 g L⁻¹ alginate, fresh, (b) 30 mmol L⁻¹ H₂O₂ and 1 g L⁻¹ alginate, fresh and (c) 10 mmol L⁻¹ H₂O₂ and 1 g L⁻¹ alginate, stored for 5 months. Size distribution was shown for the corresponding images and fitting curves were introduced as blue line.

Table 2 Mean size, size distribution and poly-dispersity index (PdI) data for the prepared nanogold colloidal solutions at room temperature, after a 30 minute reaction time

	NaOH (mmol L^−1)	Glucose (g L^−1)	H2O2 (mmol L^−1)	Alginate (g L^−1)	PdI	Size distribution (nm)	Mean size (nm)
Fresh	25	0.2	—	2.0	0.41	3.0–34.0	23.3 ± 13.1
Fresh	25	1.0	—	1.0	0.24	3.0–16.0	9.4 ± 2.7
Stored	25	1.0	—	1.0	0.46	10.0–50.0	29.0 ± 14.1
Stored	25	2.0	—	2.0	0.40	6.0–42.0	20.4 ± 10.9
Fresh	—	—	10	1.0	0.45	4.0–19.0	11.6 ± 4.6
Fresh	—	—	30	1.0	0.39	1.5–8.0	3.7 ± 1.1
Stored	—	—	10	1.0	0.58	12.0–48.0	29.6 ± 12.1
Stored	—	—	30	1.0	0.45	20.0–80.0	42.8 ± 17.3

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In the case of the H₂O₂/alginate system, the data presented in Fig. 5 show that the oxidized alginate biopolymer succeeded in the manufacture of spherical AuNPs. However, in this system, the oxidized alginate was used as a reducer for Au ions and as a stabilizer for the produced AuNPs at the same time. Smaller sized AuNPs were obtained by comparing with the alkali/glucose/alginate system. The size distribution of AuNPs was located in the range of 4–19 nm with good average size of 11.6 ± 4.6 nm (Fig. 5a and Table 2). By increasing the H₂O₂ concentration to 30 mmol L⁻¹, more reducing alginate fragments were produced in the reaction medium, permitting gold ion reduction. This explained the results in Fig. 5b and Table 2, as extremely small sized AuNPs (3.7 ± 1.1 nm) were obtained with quite a close size distribution of 1.5–8 nm. This is in support of the arguments in the interpretation of the reducing sugar content and UV-visible spectral data. After 5 months, the size distributions were expanded and the mean size was enlarged to 42.8 ± 17.3 nm (Fig. 5c and Table 2). This is logically attributed to the effect of oxidation on the role of alginate macromolecules in the stabilization of AuNPs. In spite of the storing of the nanogold colloid prepared by the H₂O₂/alginate system, it resulted in larger sized AuNPs, compared with that of the alkali/glucose/alginate system, but the stored AuNPs colloids still exhibited appropriate nano-sized dimensions.

Somehow, the size distribution presented an idea about the homogeneity in the dispersion of Au particles, but measuring the poly-dispersity index (PdI) was required for further confirmation. The PdI was investigated for the collection of Au particles in solution to determine whether there was uniformity in size and shape, and its value ranged in 0–1.⁶⁴ The AuNPs colloidal solution might have some uniformity if their size and shape are regular, as a good stable solution usually has PdI around 0.3.⁶⁵ The PdI for the so-produced AuNPs in colloidal form was measured using a zetasizer instrument and it was located in 0.24–0.58 range (Table 2). PdI values were consistent with the TEM micrographs and absorbance spectra results. Using the alkali/glucose/alginate system showed slightly better PdI than using the H₂O₂/alginate system. This confirms the stabilization of AuNPs by alginate action, which is affected by using alginate as reducer and stabilizer in the H₂O₂/alginate system. However, using glucose as reducer caused better stabilization of AuNPs, as alginate acts as a stabilizer only in the alkali/glucose/alginate system. The PdI values of 0.24 and 0.39 (close to 0.3) for the nanogold prepared by the alkali/glucose/alginate and H₂O₂/alginate systems confirm the high efficiency of the current/designed techniques to produce AuNPs with uniform size and shape. After 5 months, the PdI values were increased, reflecting the aggregation of AuNPs with time. However, the PdI values were still acceptable, confirming that the so-obtained AuNPs have an appropriate stability, even after 5 months of storage.

TEM micrographs, size distribution and PdI data all confirmed the role of the alginate biopolymer in manufacturing uniform, small sized AuNPs with good stability, using two different green systems containing alginate. Using the alkali/glucose/alginate system produced more stable AuNPs over time, while the H₂O₂/alginate system led to quite small sized AuNPs with sufficient stabilization. Comparable results were obtained by using both designed systems, but the H₂O₂/alginate system was much preferred, due to the following considerations:

• It is a totally green method, using a biocompatible/biodegradable polymer and safe oxidizing agent, H₂O₂, which is degraded to nontoxic components, O₂ and H₂O.

• It is an energy saving and costless method.

• Production of quite small sized AuNPs with sufficient stability.

• Use of a neutral medium.

The above considerations permit the use of the so-produced AuNPs, using the H₂O₂/alginate system in biomedical applications without any limitations.

XRD analysis

Nanogold prepared by using the H₂O₂/alginate system was precipitated for further characterization via XRD analysis and the data are presented in Fig. 6. Compared to the native alginate, the AuNPs–alginate composite exhibited four new diffraction peaks at scattering angles (2θ) = 38.2°, 44.7°, 64.9° and 77.8°. These new peaks correspond to the miller indices (hkl) of (111), (200), (220) and (311) face centered cubic (FCC) structure of metallic gold (JCPDS data number 04-0784 card).^66–68

Fig. 6 XRD analysis of native alginate and the AuNPs–alginate composite prepared using the H₂O₂/alginate system.

The broadening in the diffraction peaks of Au reflected the small size of the Au crystals.³⁶ The Scherrer formula (eqn (1)) was used to calculate the size of the Au crystals via analyzing the diffraction of the base peak (111) at 2θ = 38.2°. The mean size of the Au crystals was found to be in the nano dimension, 16.3 ± 1.2 nm. The marginal difference in the average size of the AuNPs between TEM micrographs and XRD data could be due to the sedimentation process of AuNPs.

where D = average particle size, K is a constant (0.9), λ is the wavelength of Cu Kα in radians, FWHM is full width at half maxima and θ is the Bragg angle.

FTIR spectra

FTIR spectroscopic analyses of native alginate and the AuNPs–alginate composite are presented in Fig. 7. For the original alginate,⁶⁹ a significant, broad absorption band at 3394 cm⁻¹ is attributed to the stretching vibrations of alginate –OH groups. The asymmetric stretching vibration of aliphatic C–H appeared at 2936 cm⁻¹. Absorption bands corresponding to the asymmetric and symmetric stretching vibrations of carboxylate groups were observed at 1616 and 1420 cm⁻¹, respectively. The structure of the original alginate from its derivatives was further characterized through the absorption bands recorded at 1044 and 942 cm⁻¹, which refer to the stretching vibration of CO of the pyranosyl ring, and CO stretching with contributions from C–C, respectively. On the other hand, some shifting in absorption bands were seen for the AuNPs–alginate, compared to the original alginate. It is clear that the absorption bands of the O–H, COO⁻ groups and CO for the pyranosyl ring were all shifted to higher wavenumbers and appeared at 3426 cm⁻¹, 1629–1430 cm⁻¹ and 1068/967 cm⁻¹, respectively. The higher shifting in wavenumbers in comparison to the original alginate is mostly attributed to the two steps suggested in the reaction mechanism (Fig. 1b); the ion exchange with Au ions and consequently, the coordination reaction between the so produced AuNPs and the referred functional groups of alginate polymeric chains. Additionally, the shifting in wave numbers is indicative of the participation of hydroxyl and carboxyl groups in coordination with AuNPs, rather than hydrogen bond formation between alginate polymeric chains.

Fig. 7 FTIR spectra of native alginate and the AuNPs–alginate composite prepared using the H₂O₂/alginate system.

4. Conclusions

The totally green synthesis of AuNPs is presented in the current work, through the use of a H₂O₂/alginate system. Alginate was used as the reducer for Au³⁺ ions and simultaneously, as the stabilizer for the so-obtained AuNPs. The role of hydrogen peroxide is the oxidative degradation of alginate at room temperature to liberate more reducible fragment moieties. The efficiency of such a system to produce nanogold was compared to that of an alkali/glucose/alginate system, in which glucose was utilized as the reducer and alginate was used as the stabilizer. The mechanism of the nanogold formation was suggested based on the understanding of the nature of the reactants. The resulting reducing sugar content in the reaction mixture was measured in both systems. FTIR spectra confirmed the suggested mechanism between alginate and Au ions in the H₂O₂/alginate system. The prepared nanogold was investigated by measuring the absorbance spectra, TEM micrographs and zetasizer results. It was found that the same reducing sugar content of 0.32 g L⁻¹ was measured by using 30 mmol L⁻¹ H₂O₂ and 1 g L⁻¹ glucose and 1 g L⁻¹ alginate. Using H₂O₂ produced AuNPs with the size distribution of 1.5–8.0 and the mean size of 3.7 ± 1.1, which is quite smaller than that manufactured in the case of glucose. XRD analysis further confirmed the formation of AuNPs and its average size was (16.3 nm) slightly higher than that measured by the zetasizer and TEM micrograph. Rapid degradation of alginate using H₂O₂ led to the production of high numbers from reducible fragments and consequently, small sized nanogold was manufactured. The poly-dispersity index was 0.39, confirming the good dispersion of nanogold. The ageing effect for 5 months showed some enlargement in the size of the nanogold to 42.8 ± 17.3 nm, which is still considered as appropriate nano dimensional size.

Compared to nanogold prepared using methods reported in literature, the so-obtained nanogold using H₂O₂/alginate can be applied in biomedical fields without any limitations, owing to the greener technique used. Additionally, the quite simple, costless and energy saving nanogold production process increases its opportunities to be widely applicable.

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