Biosynthesis of gold nanoparticles using a kind of flavonol: Dihydromyricetin

Accepted Manuscript Title: Biosynthesis of gold nanoparticles using a kind of flavonol: Dihydromyricetin Author: Qingquan Guo Qiulan Guo Juan Yuan Jinhua Zeng PII: DOI: Reference:

S0927-7757(13)00678-X http://dx.doi.org/doi:10.1016/j.colsurfa.2013.08.067 COLSUA 18638

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

16-4-2013 20-8-2013 29-8-2013

Please cite this article as: Q. Guo, Q. Guo, J. Yuan, J. Zeng, Biosynthesis of gold nanoparticles using a kind of flavonol: Dihydromyricetin, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2013), http://dx.doi.org/10.1016/j.colsurfa.2013.08.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Biosynthesis of gold nanoparticles using a kind of flavonol : Dihydromyricetin Qingquan Guo*,a, Qiulan Guoa, Juan Yuana, Jinhua Zenga School of Chemical Engineering and Light Industry, Guangdong University of

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Technology, Guangzhou 510006, China

Abstract

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The present paper investigated the biosynthesis of gold nanoparticles (AuNPs) using dihydromyricetin (DMY) without adding external surfactant, capping agent or

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template. The synthesized nanoparticles were characterized by using UV-vis spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared

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(FTIR) and particle size analyzer. TEM images showed that anisotropic growth of AuNPs happened at pH=4.5 and 5.5 and relatively uniform small spherical shaped

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gold nanoparticles were obtained at pH=6.5 and 8.5. Furthermore, both the ratios of nHAuCl4/nDMY and process temperatures were discussed respectively. DMY or DMY oxidation products which made AuNPs solution stable were directly adsorbed on the surface of AuNPs at the ratio of nHAuCl4/nDMY being 1:3 and 1:1 in the

reduction process. Besides, the monodisperse and uniform AuNPs were easily obtained at higher temperatures (90
). Based on the FTIR analysis, hydroxyl groups of DMY played an important reduction role during synthesis process. The experimental approach is simple, rapid, cost-effective and reproducible at room

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Corresponding author. Tel.: +86 13928830699;

E-mail address: [email protected]

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temperature. Keywords: biosynthesis, gold nanoparticles, dihydromyricetin, reduction mechanism,

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flavonoid 1. Introduction

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Gold nanoparticles (AuNPs) have been very important nano materials due to their

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unique and tunable surface Plasmon resonance (SPR), and used in a broad range of applications in biomedical science, such as drug delivery [1], tissue/tumor imaging [2],

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bio-labeling, biosensors devices [3], photothermal therapy [4] and electrochemical immunoassay [5]. Several existing chemical and physical methods have been reported

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to produce well-defined nanoparticles successfully. However, these processes are usually costly and involve using toxic chemicals [6-8]. In addition, the synthesis of

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AuNPs using chemical methods may lead to the presence of some toxic chemical species being adsorbed on the surface of nanoparticles, which can cause adverse

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effects in medical applications. Nowadays biosynthesis appears to be a cost efficient and more promising alternative for the preparation of gold nanoparticles due to its simplicity and eco-friendliness [9]. So far, numerous syntheses of gold nanoparticles utilizing plant extracts have been reported, such as Diospyros kaki [10], Azadirachta

indica [11], Medicago sativa [12], Aloe vera [13] and Cinnamomum camphora [14]. However, there is little information available in literature about a single substance of plant extracts to synthesize AuNPs. The synthesis of gold nanoparticles using a single active substance in plant extracts will be helpful for the purification of AuNPs and the further study of gold nanoparticles in medicine. Meanwhile, the synthesis mechanism of gold nanoparticles can be analyzed more accurately and more directly. Page 2 of 23

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At present, there are some reports that flavonoid widely existing in the plants is one of the key components which is able to reduce [AuCl4]- to gold nanoparticles [15-16]. As one type of flavonol, dihydromyricetin (DMY) has multiple biological

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effects such as hypoglycemic, antithrombotic, antioxidant, immunostimulating, anti-

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inflammatory, and antibacterial activities[17-18], etc. Its content in the vine tea (a

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plant widely distributed in southern china) can be over 20% (w/w). These factors trigger strong research interests in the field of flavonol. Hence this paper aims to

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synthesize AuNPs using DMY and investigates the influence of various reaction parameters on the morphology and size of bio-synthesized AuNPs.

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2. Experiment 2.1. Materials

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Chloroauric acid (HAuCl4·4H2O) was purchased from Sinopharm Chemical Reagent Co., Ltd (china). A stock solution of HAuCl4 was prepared by dissolving 1.0g

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HAuCl4·4H2O in 50 ml distilled water. Dihydromyricetin (≥98%) was prepared as the

best method in the literature [19] and then the stock solution (2mmol/L) of dihydromyricetin was prepared using distilled water for further experiments. All other reagents were of analytical grade with maximum purity. All glass instruments have been washed with distilled water and dried in oven before use. 2.2. Synthesis of gold nanoparticles using DMY The typical operation is that 6mL DMY solution (2mmol/L) was quickly added to 12 mL aqueous solution of HAuCl4 (1mmol/L) to generate a test sample with nHAuCl4/nDMY=1:1. Then different samples in other ratios of nHAuCl4/nDMY were produced by adjusting different amount of DMY to HAuCl4. The pH was adjusted Page 3 of 23

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with dropwise adding 10% (w/w) sodium hydroxide and the temperature was controlled using a thermostat magnetic stirrer. The reaction solution with continuously stirring became ruby red color within one minute and the color remained unchanged

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in half an hour.

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2.3. Characterization

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UV-vis spectroscopy was carried out on a TU1901 UV-vis spectrophotometer (Pgeneral, China) and the absorption maxima were analyzed at a wavelength of 400-

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900 nm. The size of particles was analyzed by using particle size analyzer, Zeta PALS (Brook havben, America), in the polystyrene cuvettes. The pH value of the solution

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was measured by PHS-3C PH (Shanghai Precision & Scientific Instrument Co., LTD,

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China). Transmission electron microscopy (TEM) studies were performed using a

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Tecnai G20 (FEI, America) electron microscope operating at an accelerating voltage of 200 kv. For the TEM measurements, a drop of the resulting solution was deposited

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on a copper grid covered with amorphous carbon. After allowing the film to stand for 2 min, the extra solution was removed with blotting paper and the grid allowed air drying before measurement. The resulting solutions were dried at 60
and then

analyzed the FTIR spectra (4000cm-1-400cm-1) on FTIR VERTEX 70 (Bruker, Germany).

3. Results and discussion 3.1 Effect of the different ratios of nHAuCl4/nDMY on the biosynthesis of AuNPs According to LaMer model, it is predicted that the formation of nanoparticles could only happen when the precursor concentration is within a suitable range for

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nucleation. However, this range might change due to different biomass-assisted synthesis approaches. We therefore studied the effect of precursor concentration representing by the different ratios of nHAuCl4/nDMY on this DMY-mediated

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synthesis. UV-vis spectroscopy was ascertained to check the formation and the

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particle size changes of AuNPs in aqueous solution. The absorption peak is assigned

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to the surface plasmon resonance (SPR) band of gold nanoparticles owing to the collective oscillation of the conduction electrons of metallic nanostructures which is

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induced by the incident light field. The intensity and width of the SPR depend on the size, morphology, spatial orientation, optical constants of the particles and the

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embedding medium [20-21]. Fig.1 showed UV-vis absorption spectra of AuNPs obtained by using a certain amount of HAuCl4 (1mmol/L) with different amount of

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DMY (2mmol/L) at different ratios of nHAuCl4/nDMY. With the ratio of nHAuCl4/nDMY increasing (from1:6 to 6:1), the symmetry of the absorption peak

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becomes increasingly clear. In Fig.2, the TEM images showed that a mixture of irregular shapes structures were formed at the ratios of nHAuCl4/nDMY being 1:3 and

1:1, while regular spherical shape was obtained at other ratios in the experiment. The number of small crystal nucleus decreased with the ratio of nHAuCl4/nDMY

increasing. The reason is possibly that excess DMY interferes with the growth of the gold nanoparticles, consequently, leading to the anisotropic growth of AuNPs and the appearance of many small nucleus. After careful observation, there are a layer of transparent membranes on the surface of the particles (Fig.2b and c). It is possibly caused by spatial orientation due to DMY or DMY oxidation product adsorbing on the surface of the gold nanoparticles. Moreover, the solutions (the ratios of Page 5 of 23

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nHAuCl4/nDMY being 1:3 and 1:1 ) were not found flocculent precipitate three months later, however, the floc emerged in other groups. Thus, we can conclude the

Fig.1 Fig.2

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3.2 Effect of the initial pH value on the biosynthesis of AuNPs

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membrane plays a very important role in the stability of the particles [22].

The initial pH value of the HAuCl4 aqueous solution is an important parameter in

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the synthesis of AuNPs using DMY. It was observed that the colors of nano gold solutions changed from nepheloid wine red to wine red and to ruby red when the

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initial pH was increased from 4.5 to 5.5, 6.5 and 8.5. Correspondingly, the UV-vis spectra (Fig.3) obtained from nano gold solutions at different initial pH values

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showed that the SPR bands of AuNPs located at different positions under different pH values. With the pH value increasing, consistently the blue shift of peak position was

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found in UV-Vis spectrum. The blue shift moved with the decreasing of the gold nanoparticles size，just as the result recorded by Zeta PALS is from 96.5 to 35.4, 24.7 and 23.8. Based on the literature, AuNPs directly synthesized by chemical methods (without templates) are generally spherical, and other morphologies are rare. Nevertheless, TEM images (Fig.4a) showed that anisotropic gold nanoparticles can be synthesized by DMY, for instance, triangles, truncated triangles, trapezoid, diamond, pentagon and hexagon morphologies. Moreover, the actual occurrence of trapezoid, diamond, pentagon and hexagon will help to guide the precise control of AuNPs anisotropic growth.. Gold is present in anionic form [AuCl4]- in acidic solution and the functional Page 6 of 23

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groups of DMY, hydroxyl groups, tend to undergo protonation and then become positively charged. The overall positively charged surface could promote the interaction between protonated hydroxyl groups and the negatively charged [AuCl4]-

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through electrostatic attraction or electrovalent bond [23]. As a result, biosorption was

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preferred over bio-reduction of [AuCl4]-. Generally speaking, the growth process

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resulting in elongation of the gold seeds rather than formation of new gold nuclei leads to the formation of anisotropic gold nanoparticles with larger dimensions[24-27].

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With the pH value increasing, at pH=5.5 (Fig.4b), the number of anisotropic AuNPs significantly was reduced. In the condition of pH=6.5 and pH=8.5, OH- could act as

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strong complexing agents of gold ions that interfere with the capping ability of DMY and also compete with the [AuCl4]- for binding to the biomolecules. So the AuNPs

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were predominantly spherical and made the anisotropic gold nanoparticles nearly

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disappeared shown in the TEM images (Fig.4c and 4d). The bio-reduction of [AuCl4]-

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could occur through the oxidation of hydroxyl to carbonyl groups as shown in Eq. (1): [ AuCl4 ]  3R  OH n  Au 0  3R  O  3nH   4Cl 

(1)

where n represents the number of hydroxyl groups in the biomolecular. DMY has six hydroxyl groups and one carbonyl group in the molecular structure which makes it have excellent reducing ability and participate in the gold bio-reduction. From the reaction stoichiometry, the redox reaction includes the transfer of three electrons and three protons, which indicates that both the kinetics and standard redox potentials could be pH dependent. The alkaline condition causes rapid reduction rate of [AuCl4]-, boosts the homogenous nucleation, and decreases in anisotropic growth. In contrast, slow reduction rate would occur under acidic condition and produce the Page 7 of 23

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heterogeneous nucleation and secondary nucleation of small Au seeds. Fig.3

3.3 Effect of temperature on the biosynthesis of AuNPs

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Fig.4

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The effect of temperature on the formation of AuNPs was also investigated. The

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color of the resulting solution remained wine red at different temperatures. The UV-vis spectra (Fig.5) showed the slight red shift of absorption wavelength and the

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particle sizes of AuNPs were 24.7, 27.8 and 43.1 respectively as the temperature increased from 30
to 60
, 90
. In addition, the shape of gold nanoparticles is

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nearly spherical as the TEM images shown (Fig.6). We can derive that the variation of particle size had a negative relationship with the reaction temperature. The reason

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could be that the process of aggregation of small AuNPs to form larger particles was favored over nuclear growth at 60
and 90
. Higher reaction temperatures usually

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resulted in a rapid reduction rate of [AuCl4]- [28] and the formation of AuNPs with

narrow size distribution. Besides, particle dispersion index (PDI) can reflect the degree of uniform particle size and is an important index of particle size characterization. The smaller PDI is, the more uniform particle size has. PDI recorded by Zeta PALS corresponded to 0.153, 0.114 and 0.062 respectively with the temperature increasing, which showed the monodisperse and uniform AuNPs were easily obtained at higher temperature (90
) which makes them have excellent application prospects. The control of uniform shape is very important in the efficient utilization of metal nanocrystals. Fig.5 Page 8 of 23

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Fig.6 3.4 Reduction mechanism Fig.7

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FTIR measurement is carried out to identify the potential functional groups

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responsible for the reduction of chloroauric acid and stabilization of the AuNPs in

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DMY. Fig.7 showed the FTIR spectra regarding DMY (before bio-reduction) and DMY+ AuNPs (after bio-reduction). The FTIR spectra revealed that four bands at

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3290cm-1, 1641cm-1, 1328cm-1 and 1168cm-1 showed significant changes after the bio-reduction. These four notable absorption peaks were assigned to several

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functional groups, such as C–O–H, C=O etc. The bands were the stretching vibration of hydroxy groups and in-plane bending vibration of hydroxy groups at 3290cm-1 and

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1328cm-1. They shifted to 3440cm-1 and 1328cm-1, respectively. Moreover, the band

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at 1168cm-1 contributed by the stretching vibration of the C-O band shifts to 1140cm-1.

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This indicated that -OH participated in the reaction. The C=O band shifted from 1641cm-1 to 1631cm-1 can be attributed to the oxidation of hydroxyl and the resulting formation of intramolecular hydrogen bonds. 4

Conclusion

The spanking rapid and simple method for biosynthesis of AuNPs by single

active substance DMY in vine tea extract offers a valuable contribution in the area of green synthesis and nanotechnology without adding toxic agent. The experiment showed that gold nanoparticles with special shapes and different sizes can be obtained by controlling the synthesis conditions accurately. During reaction process hydroxyl groups of DMY played an important reduction role. We therefore propose that the use Page 9 of 23

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of single active substance of the plant extract provides an important protocol for the exploration of the biosynthesis mechanism. In future, it would be significant to achieve better control over size, shape and absolute monodispersivity and utilize the

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potential of herbal medicine in nanoscience for anti-tumor or biomedical applications.

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Acknowledgements

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The authors acknowledge financial support of "Guangdong province 211 project program" and "the Science and Technology Project of Guangdong province "(Grant

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No. 2010B030700072)

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Figure caption Fig.1 UV-vis spectra of AuNPs obtained at different nHAuCl4/nDMY ratios (a(1:6), b(1:3) ,c(1:1),d(3:1) and e(6:1)) with pH=6.5 and T=30


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(a(1:6), b(1:3) ,c(1:1) and d(3:1) ) with pH=6.5 and T=30


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Fig.2 TEM micrographs of AuNPs obtained at different nHAuCl4/nDMY ratios

pH=8.5 ) at nHAuCl4/nDMY =6:1 and T=30


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Fig.3 UV-vis spectra recorded as a function of pH (a pH=4.5 b pH=5.5 c pH=6.5 d

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Fig.4 TEM micrographs of AuNPs obtained under different pH values of reaction solutions of a 4.5, b 5.5, c 6.5 and d 8.5 at nHAuCl4/nDMY =6:1 and T=30


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Fig.5 UV-vis spectra recorded as a function of temperature (a T=30
b T=60
c T=90
) at nHAuCl4/nDMY =6:1 and pH=6.5

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Fig.6 TEM micrographs of AuNPs obtained at different temperatures (a T=60
T=90
) at nHAuCl4/nDMY =6:1 and pH=6.5

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Fig.7 FTIR spectra of DMY before bioreduction (DMY), and after bioreduction (DMY+AuNPs) of chloroaurate acid

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Highlights • The AuNPs can be synthesized by dihydromyricetin without other additions.

• Nanometer gold solution can remain stable without additional dispersant.

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• At pH=4.5 and 5.5, we obtained the anisotropic AuNPs.

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• The synthesis of AuNPs is completed in one minute.

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*Graphical Abstract (for review)

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