Gold nanoparticles, method of preparation and applications

Abstract

Described herein are gold nanoparticles and a method of preparation. The gold nanoparticle has a hierarchical structure with an overall particle size from 40 nm to 800 nm and primary gold nanoparticles with a grain size from 4 nm to 12 nm. The gold nanoparticles may be prepared by providing an aqueous solution comprising a diblock copolymer of two hydrophilic blocks, and a water-soluble alcohol; adding a compound and a salt of Au(III) to the aqueous solution to reduce Au(III) to a Au(I)-thiolate complexes; adding a reducing agent to the Au(I)-thiolate complexes to reduce the Au(I)-thiolate complexes to primary Au(0) particles; and aggregating the primary Au(0) particles to form gold nanoparticles having a hierarchical structure.

Description

GOLD NANOPARTICLES, METHOD OF PREPARATION AND APPLICATIONS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to Singapore patent application number 10202500125S with a filing date of 15 January 2025 and titled “Double hydrophilic block copolymer-directed synthesis of complex gold nanostructures and their applications in catalysis and biosensors” and is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present disclosure relates to gold nanoparticles and methods of preparing the gold nanoparticles, and the application of the gold nanoparticles.BACKGROUND OF THE INVENTION

[0003] Gold nanostructures have gained a great deal of attentions for their usefulness in (electro)catalysis, (bio)sensing and (bio)imaging. Among various nanostructures, porous gold nanoparticles are particularly attractive due to their large surface area and abundance of low-coordination surface atom arising from its porous structure. The physiochemical properties of gold nanoparticles are highly dependent on their sizes and structures. Various methods have been developed to prepare porous gold nanostructures, such as electrochemical reduction, hard templating with preformed Au-Ag alloy nanoparticles, silver nanoparticles, or lead sulfide particles. Electrochemical reduction approaches usually give limited morphologies such as irregular powders or films on electrodes, restricting their potential applications. A hard templating method usually requires additional steps of dealloying or etching, which not only poses the concerns of contamination by remaining foreign metals but also involves harsh chemicals such as concentrated nitric acid or hydrochloric acid. On the other hand, porous gold nanoparticles may also be synthesized via a soft templating approach using surfactants or polymer as template. Porous gold nanoparticles synthesized via such approach may have more well-defined shapes and easily tunable sizes, allowing for better structure-performance correlation when in use. In addition, contamination with foreign metal can also be avoided by using gold salts as the sole metallic precursor in a bottom-up approach. Unfortunately, reports using soft templating approaches to synthesize porous gold nanoparticles are still rare to date Either a specially designed surfactant is needed, or the synthesis will involve the use of harmful (e.g., hydrazine,tetrahydrofuran), even hash chemicals (nitric acid, hydrochloric acid). It is highly desirable to develop synthesis methods using biocompatible chemicals in a more environmentally benign medium towards porous gold nanostructures.SUMMARY OF THE INVENTION

[0004] In a first aspect, there is provided a method of preparing gold nanoparticles, the method comprising providing an aqueous solution comprising a diblock copolymer of two hydrophilic blocks, and a water-soluble alcohol, wherein a ratio of a volume of water to a volume of the water-soluble alcohol is from 2.5:1 to 0.6: 1 ; adding a compound and a salt of Au(III) to the aqueous solution to reduce Au(III) to a Au(I)-thiolate complexes, the compound is selected from the group consisting of a compound of formula RXSR2, a compound of Formula MU-R^SChH, an ion of Nfb-R'-SCfH, and any combinations thereof, R1is selected from the group consisting of a Cl to C20 substituted alkyl, a Cl to C20 unsubstituted alkyl, a C6 to C20 substituted aryl, a C6 to C20 unsubstituted aryl, a C6 to C20 substituted aralkyl, and a C6 to C20 unsubstituted aralkyl, R2is selected from the group consisting of hydrogen, a Cl to CIO substituted alkyl, a Cl to CIO unsubstituted alkyl, a C6 to C20 unsubstituted aryl, a C6 to C20 substituted aralkyl, and a C6 to C20 unsubstituted aralkyl; adding a reducing agent to the Au(l)-thiolate complexes to reduce the Au(I)-thiolate complexes to primary Au(0) particles; and aggregating the primary Au(0) particles to form gold nanoparticles having a hierarchical structure.

[0005] In an embodiment, the diblock copolymer of two hydrophilic blocks consists of a first polymer block selected from the group consisting of poly(ethylene oxide), poly(vinyl alcohol), and any combinations thereof, and a second polymer block selected from poly(acrylic acid), poly(methacrylic acid), and any combinations thereof, and the aqueous solution comprises a base, preferably the first polymer block is poly(ethylene oxide) and the second polymer block is poly(acrylic acid).

[0006] Preferably, the first polymer block has a first number average molar mass (Mn) from 3,000 to 20,000, and the second polymer block has a second number average molar mass (Mn) from 1,000 to 20,000. More preferably, the diblock copolymer has a poly dispersity index from 1 to 1.5.

[0007] In an embodiment, the water-soluble alcohol is a Cl to C4 alcohol, preferably selected from the group consisting of ethanol, methanol, 1 -propanol, 2 propanol, and any combinations thereof.

[0008] In an embodiment, the compound of formula RXSR2is water soluble or is soluble in an alkaline solution (an aqueous solution with pH higher than 7 at 25°C).

[0009] In an embodiment, R1is selected from the group consisting of a Cl to CIO substituted alkyl, a C6 to CIO substituted aryl, and a C6 to CIO substituted aralkyl, wherein R1is substituted with at least one functional group selected from the group consisting of carboxylic acid, sulfonic acid, sulfinic acid, and their salts thereof.

[0010] In an embodiment, R2is hydrogen, and a molar ratio of the compound of Formula R1SR2to the salt of Au(III) is from 0.5 to less than 1.25, preferably the molar ratio of the compound of formula R1SR2to the salt of Au(III) is greater than 075 to less than 1.25.

[0011] In an embodiment, the compound of formula R'SR2is selected from the group consisting of cysteine, glutathione, homocysteine, 3 -mercaptopropionic acid, 6-mercaptohexanoic acid, 4-mercaptobenzoic acid, 3-mercaptobenzoic acid, and 2-mercaptobenzoic acid,

[0012] In an embodiment, R2is a Cl to C3 alkyl, and a second molar ratio of the compound of formula R'SR2to the salt of Au(III) is from 3 to 9, preferably the second molar ratio is from 4 to 8. More preferably, the compound of formula R1SR2is methionine.

[0013] In an embodiment, the compound is of formula NHz-R'-SChH. or the ion of NHi-R^SOsH, R1is selected from the group consisting of a Cl to CIO substituted alkyl, a C6 to CIO substituted aryl, and a C6 to CIO substituted aralkyl, preferably the compound is 2-aminoethanesulfonic acid (taurine).

[0014] In an embodiment, the salt of Au(III) is selected from the group consisting of HAuCU AuCh, and hydrates or solutions thereof.

[0015] In an embodiment, the reducing agent is selected from the group consisting of ascorbic acid, carbon monoxide, sodium borohydride, sodium cyanoborohydride, tetrakis-(hydroxymethyl)-phosphonium chloride (THPC), and any combinations thereof.

[0016] In an embodiment, the method further comprises purifying the gold nanoparticles, preferably purifying the gold nanoparticles includes at least one of the following: 1) centrifuging the gold nanoparticles, washing the gold nanoparticles, redispersing the gold nanoparticles, and repeating as necessary;; 2) treating with a mixture of sulfuric acid and hydrogen peroxide (a Piranha solution); and 3) heating at a temperature of 400°C and above.

[0017] In an embodiment, the aqueous solution consists essentially of the diblock copolymer of two hydrophilic blocks and the water-soluble alcohol, preferably wherein theaqueous solution consists of the diblock copolymer of two hydrophilic blocks and the water-soluble alcohol.

[0018] In an embodiment, the method excludes adding a metal or metal salt as a hard template to form the gold nanoparticles or the method excludes adding a non-gold metal or a non-gold metal salt.

[0019] In a second aspect, there is provided a gold nanoparticle obtained by the method according to the first aspect.

[0020] In a third aspect, there is provided a gold nanoparticle comprising a hierarchical structure with an overall particle size from 40 nm to 800 nm and primary gold nanoparticles with a grain size from 4 nm to 12 nm.

[0021] In an embodiment, the gold nanoparticle further comprises a porous structure.

[0022] Preferably, the overall particle size is formed by assembly or packing of the primary gold nanoparticles. Preferably, the overall particle size is from 80 nm to 120 nm and the primary gold nanoparticles has a grain size of approximately 5 nm.

[0023] In an embodiment, the gold nanoparticle has a bowl-like structure, the grain size of 7 nm and the overall particle size of 300 nm.

[0024] In an embodiment, the gold nanoparticle has a flower-like structure with the grain size of 10 nm and the overall particle size from 50 nm to 200 nm.

[0025] In a fourth aspect, there is provided a gold nanoparticle having the following characteristic peaks at 20 in a X-ray diffraction pattern: 38.2°, 44.5°, 64.6°, and 77.7°.

[0026] In a fifth aspect, there is provided an electrode comprising an electrode base material and the gold nanoparticle according to the second to fourth aspects or prepared by the first aspect, the gold nanoparticle is attached to the electrode base material.

[0027] In an embodiment, the electrode base material is selected from the group consisting of carbon, gold, silver, and platinum

[0028] Preferably, the electrode base material is carbon, preferably the electrode base material is selected from the group consisting of graphite, carbon black, graphene, carbon nanotubes, and combinations thereof.

[0029] In a sixth aspect, there is provided an electrochemical sensor system comprising the electrode according to the fifth aspect, a counter electrode (CE) or an auxiliary electrode (AE), and a reference electrode (RE), where the three electrodes are electrically connected when immersed in an electrolyte solution containing the analyte.

[0030] Preferably, the electrochemical sensor system is configured to operate using a differential pulse voltammetry technique or a square wave voltammetry technique.

[0031] Preferably, the electrode is configured to detect an analyte, the analyte is selected from the group consisting of a plant hormone, a drug, a drug metabolite, and combinations thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure (FIG.) 1 shows a schematic of the double hydrophilic block copolymer (DHBC) directed synthesis of porous gold nanoparticle structure in a water / ethanol mixture solution.

[0033] FIG 2A to FIG. 2H shows the characterizations of the micelles formed by DHBC in ethanol / water mixture solution and the final porous gold nanoparticle structure. FIG. 2A and FIG. 2B respectively show the transmission electron microscopy (TEM) image and dynamic light scattering (DLS) hydrodynamic diameter of DHBC micelles formed in ethanol / water mixture solution. FIG. 2C, FIG. 2D, and FIG. 2E show the TEM images of the final porous gold nanoparticles at different magnifications FIG. 2F shows the TEM image and selected area electron diffraction pattern of a single porous gold nanoparticle. FIG. 2G shows the high-angle annular dark-field scanning transmission electron microscopy (HAAD-STEM) image of the final gold nanoparticles showing a porous structure. FIG. 2H shows a typical ultraviolet-visible light (UV-vis) spectra of the porous gold nanoparticle structure.

[0034] FIG. 3 shows the role of a small thiol molecule to control the morphology of the gold nanoparticle (AuNP) structure. FIG. 3 panel A shows the molecular structure of L-cysteine. FIG. 3 panel B shows a digital image of the samples synthesized with different cysteine-to-Au ratios (0-1.25). FIG. 3 panel C shows SEM images of the particles synthesized with a cysteine-to-Au ratio of 0, 0.75 and 1.

[0035] FIG. 4 shows the different AuNP structures obtained using different small thiols during the DHBC-directed synthesis. From the top to bottom of each column, is the respective molecular structure of the thiol used, TEM image, field emission scanning electron microscopy (FESEM) image and UV-vis spectra of each AuNP structure.

[0036] FIG. 5 shows the peroxidase-like catalytic activities of various AuNPs. FIG. 5 panel A shows the UV-vis spectra taken from a mixture of 3,3',5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in solution in the absence and presence of pAuNP-1.The line in between shows the UV-vis spectrum of the mixture of pAuNP-1 and TMB only for comparison. FIG 5 panel B shows the absorbance at 650 nm (bar chart) and digital photos (inset) of the mixtures of AuNP and TMB in the presence (solid bars or photos indicated by “+”) and absence (bars with diagonal lines or photos indicated by “-”) of H2O2. FIG. 5 panel C shows a plot of initial reaction velocity for AuNP-catalyzed reaction against TMB concentration. FIG. 5 panel D shows a radar plot of the performance scores of the four AuNPs.

[0037] FIG. 6A to FIG. 6D show the electrocatalytic performance of various AuNPs. FIG. 6A shows the oxide stripping curves. FIG. 6B shows the cyclic voltammetry (CV) curves of the electrochemical oxidation of ethanol. FIG. 6C shows the summarized electrochemically active surface areas (EC SA, open squares) and specific activities (solid spheres). FIG. 6D shows the chronoamperograms of electrodes modified with various AuNPs at their corresponding peak oxidation potentials for 20 min.

[0038] FIG. 7A to FIG. 7D shows the electrochemical biosensor performance of various AuNPs. FIG. 7A shows the typical differential pulse voltammetry (DPV) curves of the AuNP modified screen-printed carbon electrodes (SPCEs) in response to indole-3-acetic acid (IAA) of varied concentrations. FIG. 7B shows the calibration curves of SPCEs modified with different AuNPs for IAA detection (n = 3). FIG. 7C shows the sensitivities of the AuNP modified SPCEs for IAA detection. Fig. 7D shows the detection of IAA in different parts of green bean sprouts.

[0039] FIG. 8 shows a mixture of DHBC solutions and ethanol at varied volume ratios.

[0040] FIG. 9 shows the mixture of DHBC solutions and ethanol at varied volume ratios after adding an equal volume (vs DHBC) of hydrogen tetrachloroaurate(III) (HAuCh).

[0041] FIG. 10 shows four reaction mixture solutions obtained using (from left to right) acetonitrile, ethanol, tetrahydrofuran and toluene as the organic solvent.

[0042] FIG. 11 shows the X-ray diffraction patterns of the four Au nanoparticle structures. The four peaks at 38.2°, 44.5°, 64.6°, and 77.7° are correlated to the (111), (200), (220), and (311) planes of gold crystals adopting a face centered cubic structure.

[0043] FIG. 12A shows the SWV curves of lysergic acid hydroxyethylamide (LSD) (0.5 mM in artificial sweat) measured on a bare SPCE (dotted line) and pAuNP-1 modified SPCE (solid line). The dash line shows the SWV curve of pAuNP-1 modified SPCE in the absence of LSD FIG. 12B shows the peak currents recorded at about 0.6V (vs Ag) for SPCEsmodified with different gold nanostructures in the presence of LSD (0.5 mM in artificial sweat).DETAILED DESCRIPTION OF THE INVENTION

[0044] In the following description, numerous specific details are set forth in order to provide a thorough understanding of various illustrative embodiments of the invention. It will be understood, however, to one skilled in the art, that embodiments of the invention may be practiced without some or all of these specific details. Embodiments described in the context of one of the methods or products are analogously valid for the other methods or products. Similarly, embodiments described in the context of a method are analogously valid for a product, and vice versa.

[0045] Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, where different elements or groups of elements are disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having a plurality of alternatives, examples of that invention with each alternative is included singly or in any combination with the other alternatives are also hereby disclosed.

[0046] Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. Where a value being discussed has inherent limits, for example where a component can be present at a concentration of from 0 to 100%, or where the pH of an aqueous solution can range from 1 to 14, those inherent limits are specifically disclosed. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention, as are ranges based thereon.

[0047] The term "alkyl" as used herein is a branched or unbranched saturated monovalent hydrocarbon radical of 1 to 24 carbon atoms (C1-C24), such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, f-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched.The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0048] Complex gold nanostructures, particularly porous gold nanoparticles have gained a great deal of attention for their wide range of applications in catalysis, electrocatalysis, surface enhanced Raman scattering (SERS), sensors, bioimaging, and biotherapy. Described herein is a soft templating method of using double hydrophilic block copolymer (DHBC)-formed micelles as the template to synthesize porous gold nanoparticle structures in environmentally benign solutions. This avoids the contamination of foreign metals and eliminates the use of harsh chemicals used in the dealloying / etching process in the hard templating method.

[0049] As illustrated in FIG. 1, the method starts by preparing an aqueous solution of double hydrophilic block copolymers made of two distinct hydrophilic blocks or monomers, such as poly(ethylene oxide)-block-poly(acrylic acid) or PEO-b-PAA in short. Then an equal volume of ethanol is added to the DHBC solution to trigger the assembly of well dispersed polymers into micelles with a PEO core and PAA shell. After that, aliquots of aqueous solutions of a small thiol molecule (R-SH, e g., L-cysteine) and gold salts (HAuC14) are added sequentially to allow for the formation of gold-thiolate oligomers in the poly(acrylic acid) (PAA) shell. An aqueous solution of ascorbic acid is then added to reduce the gold-thiolate oligomers to form tiny gold nanoclusters and induce their aggregation to form porous gold nanostructures. Finally, repeated cycles of centrifugation / re-dispersing in an ethanol / water mixture solution remove most of the polymer micelles and leave the porous gold nanoparticle structure as the final product. The harvested porous gold nanoparticles may be stored in water at room temperature for several months and redispersed by sonication upon application.

[0050] Design of double hydrophilic block copolymer-templated synthesis method for AuNP structure

[0051] Double hydrophilic block copolymers (DHBCs) are diblock copolymers consisting of two water-soluble blocks of different chemical natures. DHBCs are unique due to their aqueous solubility and adjustable amphiphilicity in response to certain substance, pH or temperature, and thus have found diverse applications such as sensors, nanoreactors, and drug delivery systems. FIG 1 shows a schematic illustrating the DHBC-directed synthesis of complex gold nanoparticles. A model DHBC, poly(ethylene oxide)-block-poly(acrylic acid) or PEO-b-PAA in short is selected and dissolved in water first. By introducing a similar amount of ethanol to the aqueous solution of PEO-b-PAA, the poly(ethylene oxide) (PEO) end becomes more hydrophobic and the DHBC tends to assemble into micelles with a PEO core and poly(acrylic acid) (PAA) shell. The formation of micelles shows the characteristic Tyndall effect (item 1, inset of FIG. 2B) while the complete solution of the DHBC in water does not (item 2, inset of FIG. 2B). The formed micelles are also confirmed by the transmission electron microscopy (TEM) (FIG 2A) and dynamic laser scattering (DLS) measurements (FIG. 2B).

[0052] The formed micelles are then used as templates in the synthesis of complex gold nanoparticle structures. As the PAA shell is abundant with carboxylic groups (COO-), direct addition of gold salts leads to rapid reduction of Au(III) to Au(0) in an uncontrolled manner as explained below and in FIG. 3. To address this issue, a small thiol molecule (R-SH), e.g., L-cysteine, is introduced first to the micelle solution. The subsequently added gold salts then react with the thiol molecule to form more stable Au(I)-thiolate oligomers. A new reducing agent, ascorbic acid, is later added to reduce the oligomers to form small gold nanoclusters. More and more gold nanoclusters are formed and then aggregated into larger particles with small gold nanoparticles as building blocks. The synthesized gold nanoparticles precipitate out of the mixture solution due to their large particle size. The collected gold nanoparticles are washed with water / ethanol (2:1 v / v) for at least thrice to remove most polymer residuals. Porous gold nanoparticles are collected as a final product (denoted as pAuNP-1).

[0053] Low magnification TEM images (FIG. 2C to 2E) show that the formed gold nanoparticles are typically spherical with an average size of about 90 nm. A high-resolution TEM image (FIG. 2F) shows the spherical gold nanoparticles adopt a hierarchical structure with small particles of approximately 5 nm as its building blocks. The small primary particles are more evident in the HA AD-STEM image (FIG 2G), which also shows the large gold nanoparticles have a porous structure. Due to the large overall size of the porous gold nanoparticle, its characteristic surface plasmon resonance (SPR) peak is observed to be located at approximately 585 nanometer (nm), which is about 65 nm red-shifted as compared to conventional gold nanospheres. FIG. 2H shows a typical ultraviolet-visible light (UV-vis) spectra of the porous gold nanoparticle structure.

[0054] FIG. 8 shows five mixture solutions of DHBC and ethanol at different volume ratios. From left to right in FIG. 8, the volume ratio of water to ethanol is 5:1, 2.5:1, 1.25:1, 0.83:1, and 0.625:1 respectively. FIG. 9 shows the five mixture solutions in FIG. 8 after anequal volume of hydrogen tetrachloroaurate(III) (HAuCh) is added. The mixtures in FIG. 9 are in the same order as in FIG. 8 When the volume ratio of water to ethanol is 2.5 or less (inclusive of 2.5), the mixture solution becomes turbid due to formation of micelles. However, when the amount of ethanol is low (e g., volume ratio of water to ethanol is 2.5 or more), the formed micelles are dissolved again after adding a solution of hydrogen tetrachloroaurate (III) (FIG. 9). When the amount of ethanol is too high, (e g., volume ratio of water to ethanol is 0.6 or less, for example when the volume ratio is 0.625 or less), aggregation of the Au(I)-thiol te complexes is very severe. Therefore, a moderate amount of ethanol (e g., volume ratio of water to ethanol is 0.83) is selected for the typical synthesis. Advantageously, the volume ratio of water to ethanol from 2.5:1 (or 2.5) to 0.6: 1 (or 0.6) is able to achieve formation of the micelles and suitable aggregation of the Au(I)-thiolate complexes.

[0055] The size and structure of Au(I)-thiolate complexes are also crucial for the formation of tiny Au (0) particles. Complexes that are too large with high polymerization degrees are hard to be reduced to form Au (0) particles. Thus, the complexes are designed to form in situ for better control of the reduction kinetics Other Au(III) salts like AuCh may also be used.

[0056] Protic water-soluble alcohols such as ethanol or methanol are most suitable to be used to prepare the gold particles. Other water miscible solvents, including polar aprotic solvents such as acetonitrile (MeCN), or nonpolar ether solvents such as tetrahydrofuran (THF), were found to be unsuccessful in synthesizing similar complex Au particle nanostructures. FIG. 10 shows images of the reaction mixture solutions using different organic solvents (from left-to-right: MeCN, ethanol, THF, toluene) with the rest of the conditions being kept the same. Amongst the tested solvent, only ethanol was able to synthesize the Au particle nanostructure, which can be visually examined by its unique color. It is believed that other water-soluble alcohols may also be used, for example Cl to C4 alcohols. Non-limiting examples include ethanol, methanol, 1-propanol, 2-propanol, and combinations thereof.

[0057] A wide range of number average molecular mass (Mn) of each block for PEO-b-PAA that are commercially available was studied and found to work as summarized below in Table 1. The number average molecular mass of the PEG block was tested from 3000 to 14000. The number average molecular mass of the PAA block was tested from 1200 to 13000. The polydispersity index (PDI) value is for the entire diblock copolymer.Polydispersity index (PDI) is defined as the ratio of the weight-average molecular weight (or weight-average molar mass) (Mr) to the number-average molecular weight (or number average molar mass) (Af„) and evaluates the monodispersity of the polymers. The PEO-b-PAA tested had a PDI from 1.1 to 1.33.

[0058] Table 1. Summary of PEO-b-PAA studied in this invention.

[0059] Other block polymers may also be used in the diblock copolymer. In an embodiment, the diblock copolymer of two hydrophilic blocks consists of a first polymer block selected from the group consisting of poly(ethylene oxide), poly(vinyl alcohol), and any combinations thereof, and a second polymer block selected from poly(acrylic acid), poly(methacrylic acid), and any combinations thereof, and the aqueous solution comprises a base, preferably the first polymer block is polyethylene oxide) and the second polymer block is poly(acrylic acid). The first polymer block may have a first number average molar mass (Mn) from 3,000 to 20,000, and the second polymer block may have a second number average molar mass (Mn) from 1,000 to 20,000. The diblock copolymer may have a poly dispersity index from 1 to 1.5.

[0060] Control of reaction kinetics by introducing small thiol molecules

[0061] The introduction of small thiol molecules is critical for the formation of complex AuNP structures via the DHBC-directed synthesis method. Thiols (-SH) are known to reduce Au' and form [Au(I)-SR]noligomers that are relatively stable. As shown in FIG. 3, the DHBC can reduce gold salts within 1 minute due to its abundant carboxylate anion (-COO-) which can donate electrons rapidly to Au 'A As a result, a gold nanoparticle (AuNP) network structure is instead formed in the absence of thiol molecules, e.g., L-cysteine (Cys), as shown in panel C of FIG. 3 where Cys = 0. After L-cysteine is introduced, the reaction kinetics of gold salts reduction is slowed down, allowing for formation of tiny primary AuNPs first and subsequent assembly into larger porous AuNP structures. An optimal L-cysteine-to-Au ratio is needed to synthesize the porous AuNP. When this ratio is small (e.g., 0.5 and 0.75), both network structure and spherical porous AuNPs may be formed. When this ratio is too large (e.g., 1.25), the Au(I)-SR bonds are hard to reduce due to the higher polymerization degreeand no AuNP structure is formed. A moderate L-cysteine-to-Au ratio (i.e., I) has been observed to best form the porous AuNP structure. The cysteine may be replaced by other thiols, thioether, or aminosulfonic acids and its ionic salt. Without being bound by theory, it is believed the key structure feature of these small molecular compounds is the sulfur atom which enables the strong soft-soft interaction of the gold and sulfur atoms. Therefore, the compound is selected from thiols, thioethers, sulfur-containing amino acids, and aminosulfonic acids etc.

[0062] The thiol or thioether may have a general formula of R'SR.2, R1is selected from the group consisting of a Cl to C20 substituted alkyl, a Cl to C20 unsubstituted alkyl, a C6 to C20 substituted aryl, a C6 to C20 unsubstituted aryl, a C6 to C20 substituted aralkyl, and a C6 to C20 unsubstituted aralkyl, R2is selected from the group consisting of hydrogen, a Cl to CIO substituted alkyl, a Cl to CIO unsubstituted alkyl, a C6 to C20 unsubstituted aryl, a C6 to C20 substituted aralkyl, and a C6 to C20 unsubstituted aralkyl. The aminosulfonic acid may have a formula of Nfb-R'-SChH. where R1is as defined above, and its ionic salt is an ion of the formula, i.e. the amine salt, sulfonate salt, or both as a zwitterion.

[0063] In an embodiment, R1is selected from the group consisting of a Cl to CIO substituted alkyl, a C6 to CIO substituted aryl, and a C6 to CIO substituted aralkyl, wherein R1is substituted with at least one functional group selected from the group consisting of carboxylic acid, sulfonic acid, sulfmic acid, and their salts thereof. In an embodiment, R2is hydrogen and R1is substituted with a carboxylic acid, i.e. the compound is a thiol with a carboxylic acid. Non-limiting examples include cysteine, glutathione, homocysteine, 3-mercaptopropionic acid, 6-mercaptohexanoic acid, 4-mercaptobenzoic acid, 3-mercaptobenzoic acid, and 2-mercaptobenzoic acid. In an embodiment, when the compound is a thiol a molar ratio of the thiol to the Au(III) salt is greater than 0.75 to less than 1.25.

[0064] In an embodiment, a thioether is used in placed of cysteine. Tn an embodiment, R2is a Cl to C3 alkyl, and a second molar ratio of the compound of formula R1SR’ to the salt of Au(III) is from 3 to 9, preferably the second molar ratio is from 4 to 8. Preferably, R1is substituted with a carboxylic acid. Non-limiting examples include methionine.

[0065] In the specific examples herein, where the thiol or thioether are amino acids or peptides, the L-amino acids are used as it is widely available, however, the D-amino acids or racemate could still be used to form the AuNPs.

[0066] In an embodiment, an aminosulfonic acid or its salt is used in place of cysteine The aminosulfonic acid may be of formula Nth-R^SChH, or the amine ion, sulfonate ionor zwitterion of NH2-R1-SOsH, R1is selected from the group consisting of a C l to C IO substituted alkyl, a C6 to CIO substituted aryl, and a C6 to CIO substituted aralkyl / In an embodiment, the compound is 2-aminoethanesulfonic acid (taurine).

[0067] Versatility of the DHBC-directed method for different AuNP structures

[0068] The DHBC-directed synthesis method is versatile in controlling the final structure of the AuNPs. FIG. 4 shows three more complex AuNP structures synthesized by replacing L-cysteine with other small thiol molecules. For instance, another porous AuNPs with an overall size of about 300 nm has been synthesized using L-m ethionine, which showed a bowl-like structure formed by small primary quasi -spherical particles and absorbs strongly from visible to near infrared regions. On the other hand, thiols such as 4-mercaptobenzoic acid or L-glutathi one lead to flower-like AuNPs with pointing spikes. Specifically, the AuNP formed with 4-mercaptobenzoic acid has an overall size of about 50 nm and exhibits an absorption peak at about 580 nm while the AuNP synthesized using L-methionine has an overall particle size of about 200 nm and exhibits a broad absorption peak at about 740-750 nm. Together with the porous AuNP formed with L-cysteine, the typical structure feature, grain size (estimated by XRD measurement) and overall sizes of four AuNP structures synthesized by introducing different thiol molecules are summarized in Table 2. In general, the four AuNP structures can be divided into two main groups according to their structural feature, i.e., porous and flower-like.

[0069] A broad range of the molecular ratio of L-methionine-to-Au (1:1 to 10:1) was studied and found that complex Au nanoparticle structures can be formed for the molecular ratio from 4: 1 to 8: 1. However, the bowl-like Au nanoparticle structure is formed best at the molecular ratio of L-methionine-to-Au of 8:1.

[0070] Table 2. Summary of different AuNP structures prepared by the disclosed DHBC-directed method.

[0071] The formed gold nanoparticles adopt a complex hierarchical structure with a large overall particle size that are fonned by assembly / packing of primary gold nanoparticles with smaller sizes into complex structures such as large raspberry-like or flower-like particles. Grain size is the size of crystallites that are small, coherent, single crystalline regions within bulkier polycrystalline materials. Grain size is usually estimated from the X-ray diffraction patterns of the solid samples. It provides valuable insights into the mechanical, optical, electronic and catalytic properties of nanomaterials.

[0072] Aliquots of solutions of the four Au nanoparticles are dropcast on a clean surface, such as a silicon wafer, and dried in air at room temperature. X-ray diffraction patterns of the dried samples are measured on a Bruker D8 X-ray diffractometer with Cu Ka radiation, 1= 1.5418 A.

[0073] The four Au nanoparticles in Table 2 show the same XRD patterns due to the face centered cubic lattice of gold crystals as shown in FIG. 11. The four AuNP structures have characteristic peaks at 38.2°, 44.5°, 64.6° , and 77.7° which are correlated to the (111), (200), (220), and (311) planes of gold crystals adopting a face centered cubic structure. However, their grain sizes are different as indicated by the difference in full width at half maximum (FWHM) of the diffraction peak. Generally, when the grain size is smaller, a broader FWHM is observed.

[0074] General procedure to synthesize sold nanoparticles (AuNPs)

[0075] In a 4-rnL glass vial containing a micro stir bar, add in sequence PEOi4.ooo-b-PAAi3.ooo (250 pL, 1.6 mg / mL in 0.25 MNaOH), L-cysteine (10 pL, 500 mM), 300 pL of anhydrous ethanol, HAuC14 (250 pL, 20 mM). The number in the subscript for the block polymer represents the number average molecular weight (Mn) of each block polymer. After two minutes of constant stirring at about 500 rpm, add in a solution of freshly prepared ascorbic acid (100 pL, 25 mM). The mixture solution changes from light yellow to greyish then black in two minutes. After another 15 minutes of constant stirring, the reaction solution is harvested and centrifuged at 12,000 rpm for 15 minutes. The precipitates are washed with ethanol / water mixture solution (Vetbanoi / Vwater = 1:2) thrice. The obtained AuNPs are redispersed in 500 pL deionized water for subsequent use and denoted as type 1 porous AuNPs (pAuNP-1)

[0076] The syntheses of the type 2 porous AuNPs (pAuNP-2), type 1 flower-like AuNPs (fAuNP-1 ), and type 2 flower-like AuNPs (fAuNP-2) are similar to that of pAuNP-1 , except that a solution of L-Methionine (160 pL, 250 mM), or L-glutathione (20 pL, 250 mM), orp-mercaptobenzoic acid (10 pL, 500 mM in IM NaOH) is used to replace L-cysteine for the synthesis of pAuNP-2, fAuNP-1, and fAuNP-2, respectively.

[0077] The gold nanoparticles may be purified by one or more of the following steps: purifying the gold nanoparticles includes at least one of the following: centrifuging the gold nanoparticles, washing the gold nanoparticles, redispersing the gold nanoparticles, and repeating as necessary; treating with a mixture of sulfuric acid and hydrogen peroxide (a Piranha solution); and heating at a temperature of 400°C and above. The steps of centrifuging, washing and redispersing the gold nanoparticles may be repeated as many times as necessary to remove unreacted reagents and impurities. Typically, the steps of centrifuging, washing and redispersing are repeated at least six times for the gold nanoparticles to be sufficiently purified and remove most if not all of the unreacted reagents and impurities.

[0078] Advantageously, the method of preparing the AuNPs involves mild conditions and biocompatible and environmentally chemicals The synthesis method is solution based, at room temperature, and fast (less than 30 minutes). The use of different small thiol molecules allows the facile control of the size and morphology of the gold nanostructures which gives different physiochemical properties.

[0079] Demonstration of peroxidase mimicking properties of the Au nanostructures

[0080] The synthesized AuNPs may be used in a variety of applications. The first application of the synthesized AuNPs structures is their nanozyme mimicking properties. FIG. 5 panel A clearly shows the peroxidase-mimi eking property of p-AuNP-1. The mixture of hydrogen peroxide (H2O2) and 3,3',5,5'-Tetramethylbenzidine (TMB) is colorless with a featureless UV absorption spectrum (lowest line in FIG. 5 panel A). However, in the presence of p-AuNP-1, the chromogenic substrate TMB changes its color to blue, indicating oxidation of TMB by H2O2 catalyzed by the peroxidase-like AuNP. As a result, the UV-vis spectrum shows a characteristic peak at about 650 nm (highest line in FIG. 5 panel A) and this peak is not due to the reaction of AuNP with TMB as shown by a control containing the AuNP and TMB (middle line in FIG. 5 panel A). Interestingly, all four AuNPs show peroxidase-like properties, as manifested by the evident blue color or absorbance change (increase in absorbance) at 650 nm of the mixtures of AuNP and TMB in the presence of H2O2 (FIG. 5 panel B). The kinetics of the peroxidase mimicking properties of these AuNPs are further studied by monitoring the initial reaction velocity against TMB concentration as shown in FIG. 5 panel C. By fitting the curve in FIG. 5 panel C with the Michaelis-Mentenequation and critical kinetic parameters such as the maximal reaction velocity (Vm), Michaelis constant (Km), catalytic constant (Kcat) and Kcat / Kmcan be derived. As shown in FIG. 5 panel D, the porous AuNPs generally show a higher maximal reaction velocity and pAuNP-2 shows the highest value. On the other hand, fAuNP-2 shows the highest affinity to the substrate TMB while pAuNP-1 shows the highest catalytic efficiency as suggested by the highest Kcat and Kcat / Km. In summary, pAuNP-1 exhibits the best peroxidase-like properties possibly due to its porous structure and overall particle size.

[0081] Electrochemical catalytic application

[0082] The electrocatalysis properties of the AuNP structures were studied using electrooxidation of ethanol as the model reaction. First, the electrochemically active surface areas (ECSA) were determined by oxide stripping in 0.5 M H2SO4 (FIG 6A). The cathodic peak at approximately 1 volt (V) is due to gold oxide stripping and used to estimate ECSA with the equation Qo= 2CNAIQ A, where Qo is the charge passed associated with oxide stripping and calculated by integration of the stripping peak. As summarized in FIG. 6C (open squares) and Table 3 below, the ECSAs of the four AuNP structures are 1.49, 0.531, 2.126 and 1.883 m2 / g, respectively. Notably, ECSA is inverse to the particle size within each type, i.e., porous or flower-type, while the 13 nm AuNP has the highest ECSA (2.3 m2 / g) due to its smallest size. Furthermore, electrocatalytic oxidation of ethanol was performed to investigate the catalytic activity and stability of various AuNPs. As shown in FIG. 6B, the characteristic peak at 0.15-0.2 V during the forward scan is attributed to the four-electron transfer of ethanol electrooxidation while the small peak at approximately 0 V during the backward scan is due to reduction of the hydroxy layer of Au and removal of the intermediate carbonaceous species formed during the electrooxidation of ethanol. The peak current density of ethanol oxidation is used to evaluate the activity of different AuNPs. After normalized to ECSA, the specific activities of the four AuNP structures are 29.34, 17.74, 11.81 and 22.35 A / m2, respectively (FIG. 6C, solid spheres, and Table 3). Notably, the specific activities of all four AuNPs are larger than that for the 13 nm AuNPs (9.9 A / m2), indicating a higher utilization efficiency of active sites for the four AuNP structures. Finally, the current decay curves measured in 1 molarity (M) ethanol and IM NaOH are used to evaluate the stability of different AuNP catalysts for electrochemical ethanol oxidation reaction (EOR). FIG. 6D shows the current densities are retained 4.3, 10.6, 4.5, and 12.7% after 20 minutes for pAuNP-1, pAuNP-2, fAuNP-1 and fAuNP-2, respectively. As a comparison, the current density retention for existing 13 nm AuNPs is only 1.5%. The resultssuggest the larger AuNP structures are more resistant to blocking of active sites due to accumulation of poisonous carbonaceous species formed in EOR.

[0083] The 13 nm AuNPs were synthesized according to an existing described protocol (Anal. Chem. 1995, 67, 4, 735-743). Briefly, an aqueous solution of sodium citrate (5 mL, 40 mM) was added rapidly to a boiling solution (100 °C) of HAuCl i (50 mL, 1 mM). The mixture was allowed to heat under reflux for 30 min to ensure complete reduction. The stirring was continued for an additional 15 min after removing the heating mantle and allowed to cool to room temperature.

[0084] Electrochemical sensors for plant hormones

[0085] The gold nanostructures may be used to detect a wide spectrum of analytes and is described further herein with specific examples, lndole-3 -acetic acid (IAA, C10EI9NO2, Molecular Weight 175.184 g / mol) is a plant hormone, which is of great importance for precision farming, crop management and plant phenotyping. FIG. 7Ato FIG. 7D shows the application of various AuNP structures obtained herein in electrochemical sensing of IAA.

[0086] The sensor is fabricated by 1) mixing equal volume of aqueous solutions of specific AuNPs and multiwall carbon nanotubes (MWCNTs) under adequate sonication and aging for 24 h to allow for aggregation / self-assembly of AuNPs on the WMCNTs, 2) drawing 5 pL of the AuNPs / MWCNTs mixture to drop-cast on the working electrodes of the SPCEs, 3) applying 4 pL of 0.5% Nafion to cover the modified working electrode and allowing it to dry in air. IAA is detected using a differential pulse voltammetry (DPV) method with a typical response shown in FIG. 7A. The anodic peak at approximately 0.6V is due to electrooxidation of IAA and the peak current is used to estimate the amount of IAA. The calibration curves of IAA sensors modified with various AuNPs are shown in FIG. 7B. The slope of the linear region is used to calculate the sensitivities to evaluate the efficiencies of the sensor.

[0087] As shown in FIG. 7C and Table 3, fAuNP-2 shows the highest normalized sensitivity of 76.2 pA / rnM • mM-1Au, possibly due to its flower-like structure which exposes more active sites at its multiple pods. fAuNP-2 is further used for IAA sensing in real plants due to its highest sensitivity. Green bean sprouts after four days of growth have been selected as a model plant It has been found that the concentration of IAA is 0.027, 0.225, 0.093 mM in the bud, stem and root, respectively (FIG. 7D). The observed values are consistent with the reported ones for both bud and stem while higher than literature for the root part.

[0088] Table 3. Comparison of AuNPs prepared by the methods herein and existing AuNP

[0089] Electrochemical sensors for illicit drugs

[0090] It was found that the gold nanostructures can enhance the signal readout when they are used as a lysergic acid hydroxyethylamide (LSD) sensor. The LSD sensor was functionalized by dropcasting a small drop of the respective gold nanostructure in aqueous solution on the electrode surface of a screen-printed carbon electrode and used to detect LSD via a square wave voltammetry (SWV) method. A typical example of the square wave voltammetry measurement is shown in FIG. 12A, the peak current at 0.6 V (vs Ag) arising from LSD can be boosted from 10 pA for the bare electrode to 19 LLA for the electrode modified with pAuNP-1 which is a 90% enhancement of the signal strength. Notably, all four types of gold nanostructures could boost the signal of LSD with an enhancement factor of 50-100% as shown in FIG. 12B.

[0091] The methods described herein allow for the preparation of gold nanoparticles with a distinct and unique structure. The gold nanoparticles may be used in a wide variety of applications including as a catalyst in electrochemical applications for example in fuel cells involving electrocatalytic oxidation of alcohols, and as a sensor to detect a wide variety of analytes. Non-limiting examples of sensors that may use the gold nanoparticles include colorimetric or fluorometric enzyme-based sensors, plant healthcare sensors for growth monitoring, disease managing, etc., surface-enhanced Raman spectroscopy (SERS)-based sensors for food security, and electrochemical-based wearable sensors for healthcare and lifestyle. Specific examples of the analyte that may be detected includes plant hormones like IAA and illicit drugs like LSD.

[0092] As it may be seen in the various applications of the various AuNPs prepared by the methods herein, different AuNP structures are the most suitable for each application. The facile control of the size and morphology by changing thiol molecule in the synthesis provides a versatile and facile method of preparing different AuNP for the required application.

Claims

Claims1 A method of preparing gold nanoparticles, the method comprisingproviding an aqueous solution comprising a diblock copolymer of two hydrophilic blocks, and a water-soluble alcohol, wherein a ratio of a volume of water to a volume of the water-soluble alcohol is from 2.5:1 to 0.6:1;adding a compound and a salt of Au(III) to the aqueous solution to reduce Au(III) to a Au(I)-thiolate complexes,the compound is selected from the group consisting of a compound of formula RXSR2, a compound of Formula NF -l -SOsH, an ion of NFh-l -SOsH, and any combinations thereof, R1is selected from the group consisting of a Cl to C20 substituted alkyl, a Cl to C20 unsubstituted alkyl, a C6 to C20 substituted aryl, a C6 to C20 unsubstituted aryl, a C6 to C20 substituted aralkyl, and a C6 to C20 unsubstituted aralkyl, R2is selected from the group consisting of hydrogen, a Cl to CIO substituted alkyl, a Cl to CIO unsubstituted alkyl, a C6 to C20 unsubstituted aryl, a C6 to C20 substituted aralkyl, and a C6 to C20 unsubstituted aralkyl, adding a reducing agent to the Au(T)-thiolate complexes to reduce the Au(I)-thiolate complexes to primary Au(0) particles; andaggregating the primary Au(0) particles to form gold nanoparticles having a hierarchical structure.

2. The method according to claim 1, wherein the diblock copolymer of two hydrophilic blocks consists of a first polymer block selected from the group consisting of poly(ethylene oxide), poly(vinyl alcohol), and any combinations thereof, and a second polymer block selected from poly(acrylic acid), poly(methacrylic acid), and any combinations thereof, and the aqueous solution comprises a base, preferably the first polymer block is polyethylene oxide) and the second polymer block is poly(acrylic acid).

3. The method according to claim 2, wherein the first polymer block has a first number average molar mass (Mn) from 3,000 to 20,000, and the second polymer block has a second number average molar mass (Mn) from 1,000 to 20,000.

4. The method according to claim 3, wherein the diblock copolymer has a polydispersity index from 1 to 1.5.

5. The method according to any one of claims 1 to 4, wherein the water-soluble alcohol is a Cl to C4 alcohol, preferably selected from the group consisting of ethanol, methanol, 1 -propanol, 2 propanol, and any combinations thereof.

6. The method according to any one of claims 1 to 5, wherein the compound of formula R'SR2i s water soluble or is soluble in an alkaline solution (an aqueous solution with pH higher than 7 at 25°C).

7. The method according to any one of claims 1 to 6, wherein R1is selected from the group consisting of a Cl to CIO substituted alkyl, a C6 to CIO substituted aryl, and a C6 to CIO substituted aralkyl, wherein R1is substituted with at least one functional group selected from the group consisting of carboxylic acid, sulfonic acid, sulfmic acid, and their salts thereof.

8. The method according to any one of claims 1 to 7, wherein R2is hydrogen, and a molar ratio of the compound of Formula R'SR2to the salt of Au(III) is from 0.5 to less than 1.25, preferably the molar ratio of the compound of formula R1SR2to the salt of Au(III) is greater than 0.75 to less than 1.25.

9. The method according to claim 8, wherein the compound of formula RXSR2is selected from the group consisting of cysteine, glutathione, homocysteine, 3- mercaptopropionic acid, 6-mercaptohexanoic acid, 4-mercaptobenzoic acid, 3- mercaptobenzoic acid, and 2-mercaptobenzoic acid,10. The method according to any one of claims 1 to 7, wherein R2is a Cl to C3 alkyl, and a second molar ratio of the compound of formula R'SR2to the salt of Au(III) is from 3 to 9, preferably the second molar ratio is from 4 to 8.

11. The method according to claim 10, wherein the compound of formula RLSR2is methionine.

12. The method according to any one of claims 1 to 6, wherein the compound is of formula NH2-R1-S0sH, or the ion of NH2-R1-SOsH, R1is selected from the group consisting of a Cl to C 10 substituted alkyl, a C6 to CIO substituted aryl, and a C6 to CIO substituted aralkyl, preferably the compound is 2-aminoethanesulfonic acid (taurine).

13. The method according to any one of claims 1 to 12, wherein the salt of Au(III) is selected from the group consisting of HAuCh, AuCh, and hydrates or solutions thereof.

14. The method according to any one of claims 1 to 13, wherein the reducing agent is selected from the group consisting of ascorbic acid, carbon monoxide, sodium borohydride, sodium cyanoborohydride, tetrakis-(hydroxymethyl)-phosphonium chloride (THPC), and any combinations thereof.

15. The method according to any one of claims 1 to 14 further comprising purifying the gold nanoparticles, preferably purifying the gold nanoparticles includes at least one of the following: 1) centrifuging the gold nanoparticles, washing the gold nanoparticles, redispersing the gold nanoparticles, and repeating as necessary;; 2) treating with a mixture of sulfuric acid and hydrogen peroxide (a Piranha solution); and 3) heating at a temperature of 400°C and above.

16. The method according to any one of claims 1 to 15, wherein the aqueous solution consists essentially of the diblock copolymer of two hydrophilic blocks and the water-soluble alcohol, preferably wherein the aqueous solution consists of the diblock copolymer of two hydrophilic blocks and the water-soluble alcohol.

17. The method according to any one of claims 1 to 16, wherein the method excludes adding a metal or metal salt as a hard template to form the gold nanoparticles or the method excludes adding a non-gold metal or a non-gold metal salt.

18. A gold nanoparticle obtained by the method according to any one of claims 1 to 17.

19. A gold nanoparticle comprising a hierarchical structure with an overall particle size from 40 nm to 800 nm and primary gold nanoparticles with a grain size from 4 nm to 12 nm.

20. The gold nanoparticle according to claim 19 further comprising a porous structure.

21. The gold nanoparticle according to claim 19 or claim 20 wherein the overall particle size is formed by assembly or packing of the primary gold nanoparticles.

22. The gold nanoparticle according to any one of claims 19 to 21, wherein the overall particle size is from 80 nm to 120 nm and the primary gold nanoparticles has a grain size of approximately 5 nm.

23. The gold nanoparticle according to claim 20 with a bowl-like structure, the grain size of 7 nm and the overall particle size of 300 nm.

24. The gold nanoparticle according to claim 19 with a flower-like structure with the grain size of 10 nm and the overall particle size from 50 nm to 200 nm.

25. A gold nanoparticle having the following characteristic peaks at 20 in a X-ray diffraction pattern: 38.2°, 44.5°, 64.6°, and 77.7°.

26. An electrode comprising an electrode base material and the gold nanoparticle according to any one of claims 18 to 25 or prepared by the method according to any one of claims 1 to 17, the gold nanoparticle is attached to the electrode base material.

27. The electrode according to claim 26, wherein the electrode base material is selected from the group consisting of carbon, gold, silver, and platinum.

28. The electrode according to claim 27, wherein the electrode base material is carbon, preferably the electrode base material is selected from the group consisting of graphite, carbon black, graphene, carbon nanotubes, and combinations thereof.

29. An electrochemical sensor system comprising the electrode according to any one of claims 26 to 28, a counter electrode (CE) or an auxiliary electrode (AE), and a reference electrode (RE), where the three electrodes are electrically connected when immersed in an electrolyte solution containing the analyte.

30. The electrochemical sensor system according to claim 29, wherein the electrochemical sensor system is configured to operate using a differential pulse voltammetry technique or a square wave voltammetry technique.

31. The electrochemical sensor system according to claim 29 or 30, wherein the electrode is configured to detect an analyte, the analyte is selected from the group consisting of a plant hormone, a drug, a drug metabolite, and combinations thereof.

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