Green synthesis of gold nanoparticles and their emerging applications in cancer imaging and therapy: a review

2.1 Physical methods

The ultrasonic spray pyrolysis (USP) technique is one of the common physical methods that has gained strong potential for large-scale production of AuNPs. ⁴² The synthesis of AuNPs using USP is straightforward and versatile, and the functional properties of the NPs can be tuned by changing the process parameters. ⁴³ Because of operating at ambient conditions (i.e., room temperature and atmospheric pressure), which consumes less energy, USP is considered more affordable than other physical processes such as ball milling, ultrasonic machining, laser ablation. ⁴⁴ A USP device is composed of an ultrasonic generator, a reaction chamber, and a collection medium, which typically serve as a stabilizing agent to prevent/minimize agglomeration. ^{42 , 45} For running a USP process, the droplets of Au precursor solution are first generated by the ultrasonic generator. These droplet-like aerosol particles are then passed into the heating chamber using an inert carrier gas. After the complete chemical reactions at extremely high temperatures, the droplets containing AuNPs are then collected ⁴² (Figure 1). Bekić at el. ⁴⁶ reported the synthesis of AuNPs through the USP technique using hydrogen tetrachloroaurate (III) hydrate (HAuCl₄·3H₂O) as the precursor. The precursor was first subjected to an ultrasonic aerosol generator at a frequency of 2.5 MHz to form aerosol droplets of the solution. A nitrogen carrier gas at the flow range of 1–4.5 L/min then transported the aerosol droplets to the heating zones via a quartz tube. To generate pure AuNPs, hydrogen gas (H₂) was added as the reducing agent to the flowing nitrogen gas. At the first heating zone (referred to as evaporation zone), the temperature was set in the range of 50−100 °C to evaporate and dry the aerosol particles. At the second heating zone, the drying temperature increased to 260−500 °C to enable the formation of AuNPs. After completing the reaction, the AuNPs were collected in a separate collection medium containing sodium citrate solution as a stabilizer. The observation of AuNPs particles using high-resolution TEM and SEM revealed an approximate size of 20 nm and spherical or polyhedron shapes. A characterization using UV–vis spectroscopy, the surface plasmon resonance (SPR) peak was found to be located at the wavelength of 530 nm, the conclusion drawn from the wavelength shift from 530 nm to 532 nm after coating AuNPs with PVP and PEG suggests that the increase in hydrodynamic size and the shift in the SPR peak is likely attributed to the coating of AuNPs with larger polymers, PVP and PEG. This conclusion indicates that the coating process alters the optical properties of the AuNPs, affecting their interaction with light. Similarly, Golub et al. ⁴² dissolved HAuCl₄ salt in deionized water and magnetically stirred the mixture until its color turned a bright yellow. This yellowish solution was used as the precursor for the synthesis of AuNPs. The authors then used an ultrasonic nebulizer, which was operated at a frequency of 2.5 MHz to generate submicron aerosol droplets. The aerosols were then transferred to the heating zones via a quartz glass tube using nitrogen as the carrier gas (6 L/min). Hydrogen gas (3 L/min) was mixed with the gas inlet for the reduction of the aerosol droplets to AuNPs. The heating region was designed to have four different zones: one evaporation zone, which separates the aerosol droplets, and three drying zones, which dry the particles. The temperature in the evaporation zone was 120 °C, while the temperature in three consecutive reaction zones was 350 °C to allow the synthesis of AuNPs. The polyvinylpyrrolidone (PVP)-coated AuNPs with a nominal size of 28.04 nm were then collected in a collection medium. The colloidal AuNPs remained stable with the presence of SPR at 532 nm.

Figure 1:

A schematic presentation of an ultrasonic spray pyrolysis (USP) technique that is composed of an ultrasonic nebulizer, a four-zone heating region, collection bottles, a gas inlet and outlet, and a quartz transport tube. Adapted from ref. 42].

Laser ablation (LA) is another physical method that removes NPs from a target metal surface. During laser ablation, a high-energy pulsed laser beam is irradiated on top of the metal solid surface. Ablation (vaporization and ejection of fragments) only occurs when the metal surface absorbs sufficient energy. ⁴⁷ Laser ablation in liquid (LAL) has been investigated by many groups to enhance the efficiency of the laser greatly. Figure 2a depicts a schematic illustration of a typical LAL system that is often used for synthesizing AuNPs. The ablation of a bulk material immersed in a liquid prevents the re-deposition of ablated NPs on the material’s surface. The liquid environment also absorbs the heat of the metal target, preventing excessive heat build-up. ³¹ Shamari et al. ⁴⁸ devised an ablation apparatus that used highly pure Au targets in their experiment. Before ablation, the target was sanded and sonicated in an organic solvent. The Au was then washed with chlorobenzene and distilled water several times to eliminate any possible impurities on the surface. The ablation was performed using a high-energy Nd:YAG laser that operated at a pulse frequency of 10 Hz, an emission wavelength of 1,064 nm, a width pulse of 6 nm, and a laser power of 4 W. A convex lens (focal length = 10 cm) was used to guide the laser beam to focus perpendicularly onto the target surface. The Au target was submerged in water before being irradiated with a laser beam. The X-ray diffraction (XRD) and UV–vis spectroscopy results collectively validated the production of crystalline AuNPs with the face-centered cubic crystal structure. The TEM imaging revealed the average diameter of the spherical AuNPs to be around 10–25 nm. In another study, Menazea et al. ⁴⁹ obtained AuNPs with a diameter of 20 nm through an ablation process that lasted only 10 min. The authors used an Au plate with 99.99 % purity, which had been polished repeatedly using ultra-fine emery paper, followed by washing in water. The process used a mixture of chitosan (CS) and polyvinyl alcohol (PVA) in a ratio of 7:3 ratio as the liquid medium. An Nd:YAG laser with a pulse frequency of 10 Hz, a basic wavelength of 1,064 nm, a pulse duration of 7 ns, and an output power of 3.6 W was used for the ablation process. The metal plate was partially submerged inside a beaker containing 20 mL of the CS/PVA blend. To focus the laser beam on the metal target, the plate was moved in a direction perpendicular to the laser beam using a convex lens. The TEM images showed the fragmented spherical AuNPs scattered in CS/PVA solution with sizes of roughly 20 nm (Figure 2b). In summary, the liquid medium used in laser ablation, whether water or a CS/PVA blend, affects the temperature and chemical environment during the process, impacting the shape, size, and dispersion of synthesized AuNPs. Water assists in heat absorption and inhibits re-deposition, resulting in crystalline AuNPs with an average diameter of 10–25 nm, whereas the CS/PVA blend produces fragmented AuNPs scattered in solution, each around 20 nm in size. Further research into the unique impacts of various liquid media is required for a complete knowledge of laser ablation synthesis.

Figure 2:

LAL synthesis AgNPs. (a) Schematic illustration of a typical water-submerged ablation system which is commonly utilized for synthesizing AuNPs ⁴⁸ and (b) TEM images of AuNPs generated using a submerged laser ablation system using CS/PVA as the liquid medium. Adapted from ref. 49].

2.2 Chemical methods

The Turkevich method has long been considered the most well-known technique to synthesize colloidal AuNPs due to its simplicity, controllable size, and ease of synthesis. ^{7 , 50} This method uses sodium citrate as a reducing agent to reduce Au³⁺ ions in a heated environment. ⁷ The Turkevich method was used by Mintz et al. ⁵¹ to synthesize AuNPs with an average size of ∼23 nm shown by AFM and TEM measurements (Figure 3). To synthesize AuNPs using the Turkevich method, a solution of sodium citrate is added to boiling aqueous solution of chloroauric acid (HAuCl₄) and mixture is vigorously stirred. A few minutes after stirring under boiling temperature, the mixture is cooled down in the dark. Like physical methods, AuNPs synthesized in a chemical manner had an absorbance peak of 525 nm. Using the concept presented by the Turkevich method, Sangamithirai et al. ⁵² achieved spherical AuNPs with a diameter of ∼5 nm_. HAuCl₄ aqueous solution was heated to boiling in a flask with continuous stirring, followed by the addition of sodium citrate. The mixture was then stirred for an additional 15 min under the same boiling setting until its color changed from pale yellow to red. The resulting citrate-stabilized AuNPs colloidal solution was slowly cooled to room temperature. The crystal patterns of AuNPs, identified using XRD, aligned with the JCPDS card no. 04-0784, ⁵³ confirming the face-centered cubic polycrystalline structure. The crystallite size, calculated via the Scherrer equation, was approximately 5 nm, closely matching the size observed in high-resolution TEM images. Previous studies by Mintz et al. and Sangamithirai et al. utilized the Turkevich method with sodium citrate as a reducing agent. Sangamithirai et al., who preheated the HAuCl₄ solution, achieved smaller AuNPs (∼5 nm) compared to Mintz et al.’s results (∼23 nm). This approach likely optimized nucleation and growth dynamics, producing smaller and more uniform AuNPs.

Figure 3:

TEM image of AuNPs synthesized through Turkevich method (a); TEM-derived size distribution histogram showing an average diameter of ∼ 23 nm (b); AFM image of the synthesized AuNPs through Turkevich method (c); and height profile generated from AFM image (d). Reprinted from ref. 51].

Recent efforts have simplified the Turkevich method. For instance, it was found that incorporating sodium borohydride (NaBH₄) into the protocol, either with or without sodium citrate, can eliminate the heating step. ³⁵ Utilizing this adaptation, Wang et al. ⁵⁴ synthesized AuNPs with a diameter of 4 nm by adding sodium citrate to an HAuCl₄ solution at room temperature, followed by the addition of ice-chilled NaBH₄. The solution turned from colorless to red, indicating the formation of AuNPs, and, after stirring for 30 min, was left undisturbed for 2 h. The UV–vis spectrum showed a predominant SPR peak at 509 nm, with DLS indicating a hydrodynamic size of 6.3 nm and TEM calculating it at 5.0 nm. Following a similar method, Chaudhary et al. ⁵⁵ synthesized AuNPs (7.53 nm) by mixing HAuCl₄ and sodium citrate solutions at room temperature, then adding NaBH₄ dropwise. The reddish color of the solution confirmed AuNP formation, and residual NaBH₄ was decomposed by stirring for 24 h. XRD and EDX analyses verified the face-centered cubic configuration of AuNPs, while Zeta potential measurements (∼30 mV) indicated good stability.

The Brust-Schiffrin method is another well-known technique to produce spherical AuNPs that are soluble in organic solvents. ³⁵ The Brust-Schiffrin method is a two-phase process: (i) a phase transfer agent is used to transfer Au³⁺ ions from an aqueous-based to an organic-based phase. (ii) The organic-based Au³⁺ ions are then reduced through alkanethiol-mediated NaBH₄. ⁵⁶ This method produces small-sized NPs (<10 nm) due to the strong affinity of thiol ligands on the Au surface, which prevents the self-assembly and aggregation of AuNPs. ³⁵ Quintana et al. ⁵⁷ combined aqueous solution of HAuCl₄ with tetraoctylammonium bromide (TOAB) – toluene as a new strategy to synthesize AuNPs. Dodecanethiol which served as a stabilizer, was then added to the mixture, and AuCl⁴⁻ ions were reduced to AuNPs upon dropwise addition of NaBH₄. The organic solvent changed the color of the solution from orange to dark brown, implying the formation of AuNPs. The toluene (organic) phase was then removed from the water phase after further stirring for 30 min. The toluene phase was concentrated before being diluted with ethanol at the ratio of 1:200. The solution was kept at 22 °C for 2 h and was finally filtered through a nylon membrane and rinsed with ethanol. The TEM observation demonstrated tiny particles with an average size of 1.8 nm. In another study, Lee et al. ⁵⁸ successfully synthesized 5-nm AuNPs by mixing aqueous HAuCl₄ with TOAB-toluene solution. After removing the aqueous phase, NaBH₄ was gradually added to the toluene phase. After stirring the mixture for 24 h, the toluene phase was collected and rinsed with sulfuric acid followed by deionized water. The aqueous solution was further stirred with an excess amount of dodecanethiol. The evaporation of toluene in the solution resulted in dried powder. To remove the unreacted reactants from AuNPs, the powder was uniformly distributed in chloroform and centrifuged at 25,000 rpm for 10 min. The powdered precipitate was then re-suspended in chloroform and stored at 4 °C.

The sonochemical method is a facile and rapid technique to obtain AuNPs. The operational mechanism of sonochemical method is to use a high-intensity ultrasound to molecules to initiate acoustic cavitation-induced chemical reactions. ^{59 , 60} When a liquid is exposed to ultrasound radiation, the periodic expansion and compression yield high- and low-pressure zones. ⁶¹ The presence of singular behavior in the liquid medium causes acoustic cavitation, which is the formation, expansion, and collapse of microbubbles. ⁵⁹ Figure 4a illustrates the process of acoustic cavitation driven by ultrasonic radiation. After bubbles collapse, the pressure difference between the inner and outer sections of bubbles produces a shock wave. ^{62 , 63} In this context, ultrasonic waves cause better mechanical stirring than normal stirring. ⁶⁴ The mechanical stirring, as along with the formation of high temperature and pressure caused by shock wave, collectively accelerate the radical formation and reduction of Au precursor. ^{64 , 65} There are many efforts dedicated to using ultrasound radiation to synthesize AuNPs. For example, Dheyab et al. ⁹ performed the sonochemical-assisted synthesis of AuNPs using the combined aqueous solution of HAuCl₄ and sodium citrate as precursors. The authors conducted the experiment by slowly adding 2 mL of 0.03 M HAuCl₄ solution into 20 mL of aqueous solution of 0.03 M sodium citrate. Sonication at a low frequency of 20 kHz and three different sonication powers (12, 20, and 36 W) were performed for 10 min to achieve complete mixing. XRD scans exhibited a single-face-centered cubic Au phase with high crystallinity. TEM size distribution measurements determined the diameters of spherical AuNPs to be 22.3, 18.6, and 13.6 nm for 12, 20, and 36 W power, respectively. With the increase of ultrasound power from 12 W to 36 W, the SPR peak shifted from 518 nm to 523 nm. The shift in the peak position of the UV–vis spectra from 518 nm to 523 nm indicates a redshift, which suggests an increase in particle size. In this context, it’s a positive outcome as it correlates with enhanced catalytic activity, with the most effective catalysis observed when the gold nanoparticles were approximately 13.6 nm in size. The zeta potential was found to be strongly dependent on sonication power. The zeta potentials of AuNPs were measured to be −35.7, −36.1, and −37.1 mV when sonication was conducted at an output power of 12, 20, and 36 W, respectively. These results suggested that increasing the sonication power from 12 W to 36 W led to much negative zeta potential and better water stability.

Figure 4:

Chemical method synthesis AuNPs. (a) The process of the ultrasonic radiation that is used to generate acoustic signals for the synthesis of AuNPs. Reprinted from ref, 59]. (b) TEM image of synthesized Au nanodumbbells; and (c) UV–vis spectral analysis of Au nanodumbbells, indicating the size-dependent optical properties of AuNPs. Adapted from ref. 66].

The seeding-growth method is another method to synthesize rod-shaped AuNPs. The reduction of an Au precursor governed by a reducing agent is performed to produce Au seed particles. Then, the single-crystal spherical Au seeds are grown into rod-like shape nanoparticles through a seed-mediated growth strategy. ^{35 , 50} Meng et al. ⁶⁶ used this method to synthesize Au nanodumbbells from Au nanorods. HAuCl₄ as Au precursor reacted to cetyltrimethylammonium bromide (CTAB) solution at room temperature, followed by introducing ice–cold NaBH₄ solution, which turned the solution into a yellowish-brown color. The prepared seed solution was continuously stirred at 27 °C for 2 min to form a growth solution. The growth of Au nanorods can be controlled by the addition of CTAB to silver nitrate (AgNO₃) solution at room temperature. Next, HAuCl₄, nitric acid (HNO₃), and ascorbic acid were infused into the growth solution. The solution turned colorless from dark yellow. The seed solution was finally converted into the growth solution, creating Au nanorods with an aspect ratio of ∼2.8. The Au nanorods were collected by centrifuging the colloidal solution at 8,000 rpm for 10 min for two cycles, discarding the supernatant liquid, and redispersing it in fresh CTAB solution. The growth solution of Au nanodumbbell was prepared by adding HAuCl₄ into CTAB and vigorously stirring at room temperature, followed by the addition of ascorbic acid. The color of the growth solution changed from yellow to colorless. The synthesis was concluded by adding nanorod seeds into the growth solution, producing Au nanodumbbells with a size of 86 nm in length, as seen from the TEM image (Figure 4b). The UV–vis spectral analysis revealed a broad SPR band at 785 nm and a lower absorption band around 530 nm due to the spherical AuNPs impurities and surface plasmons traveling across the transverse axis, respectively (Figure 4c). Nayef et al. ⁶⁷ claimed that AuNPs of increasing sizes could be obtained with the increase in the volume of seed solution. Sodium citrate was included in the HAuCl₄ solution, followed by ice–cold NaBH₄. After 5 min of stirring, the seed solution was allowed to rest for 1 h at room temperature. The growth solution was made by combining HAuCl₄, CTAB, and ascorbic acid. Adding increasing amounts of seed solution (1 mL, 3 mL, and 10 mL) to the growth solution generated AuNPs of increasing sizes, which were 31.41 nm, 32.38 nm, and 47.86 nm, respectively (Table 1).

3.1 Green synthesis of AuNPs using bacteria

The bacterial biosynthesis of AuNPs could take place intracellularly or extracellularly where they are formed. ³⁷ With the secretion of enzymes from bacteria, Au ions are enzymatically reduced to AuNPs. The enzyme-mediated reduction seems to be among the most convenient and environmentally friendly routes for synthesizing AuNPs. ²⁷ The nitrate reductase is a typical enzyme that is crucial in the reduction of Au ions. ^{68 , 69} In the synthesis of AuNPs through the extracellular route, the surface-trapped Au ions are reduced with the presence of enzymes on the cell wall, whereas in the intracellular synthesis pathway, the Au ions are diffused into the cell that contains enzymes for their efficient reduction to AuNPs ³¹ (Figure 5).

Figure 5:

Schematic illustration of bacterial synthesis of AuNPs through intracellular and extracellular pathways. This figure demonstrates enzyme-driven reduction processes in bacterial synthesis of AuNPs, showing potential for environmentally friendly synthesis. Adapted from ref. 70].

The extracellular synthesis of AuNPs has attracted greater interest than intracellular synthesis because of the demand for fewer steps to release NPs. ¹³ Besides, AuNPs that are produced outside of cells through extracellular pathways do not have attached cellular components and, thus, can be used without purification for a variety of biomedical applications. The strengths of using bacteria for the synthesis of AuNPs lie in its simplicity of handling/operation, low cost, and efficiency. ⁷¹ However, despite the benefits, this technique has some drawbacks such as the inadequacy of control over the morphology and size of the AuNPs. ¹³ The underlying principles and molecular-level mechanisms to synthesize AuNPs using bacteria also are largely unknown. Several studies have attempted to understand these mechanisms at molecular levels to better control the size, shape, and crystallinity of AuNPs. For example, Patil et al. ⁷² managed to synthesize AuNPs with an average size of ∼21 nm by using extracellular metabolites of Paracoccus haeundaensis BC74171^T, which is a marine bacterium. After incubation, the culture was centrifuged at 3,500 rpm for 30 min to separate the cells. HAuCl₄ and the collected supernatant were mixed and stirred magnetically in a heated water bath at 70 °C for 15 min, leading to the formation of AuNPs. The synthesized AuNPs were centrifuged, washed with distilled water, and finally freeze-dried to obtain powder form. The authors have shown the formation of AuNPs through XRD and EDX analyses. TEM observation revealed that the size of spherical AuNPs ranged between 15 and 35 nm. An SPR peak at 534 nm was found in the UV–vis spectrum, while the zeta potential of AuNPs was measured to be 4.26 mV. In another study, Markus et al. ⁷³ used a probiotic isolated from Korean kimchi, Lactobacillus kimchicus DCY51^T, to intracellularly synthesize spherical-shaped AuNPs with average particle sizes ranging from 5 nm to 30 nm (Figure 6). After sequential incubation and centrifugation, the collected cells were suspended in sterile water Followed by adding AuCl₄H₃O into the suspension. The suspension was kept at 30 °C in the darkness. Visual examination for color change of the suspension was used to monitor the completeness of the AuNPs synthesis. The intracellularly synthesized AuNPs suspension was then centrifuged and underwent ultra-sonication several times before being rinsed with 80 % methanol to remove any unwanted or unreacted components. The powder form of AuNPs was finally collected by air-drying. The UV–vis showed an absorbance peak at ∼540 nm. EDX analysis showed the presence of metallic Au as the major element in the sample, whereas XRD and SAED (Selected Area Electron Diffraction) confirmed the crystalline nature of the synthesized AuNPs.

Figure 6:

Formation of AuNPs through intracellular synthesis using Lactobacillus kimchicus DCY51^T, showing the role of bacterial processes in shaping AuNP size and morphology. Adapted from ref. 73].

3.2 Green synthesis of AuNPs using plant extracts

Plant species are an inexhaustible source of medicinal substances and have long been used for many medical practices. ^{37 , 74} As such, they have the potential to be used as a green-reducing agent for synthesizing AuNPs. Compared to bacteria-mediated synthesis, plant-mediated AuNP synthesis has a number of benefits, including the greater upscale potential by removing the time-consuming and costly stages of cell culturing. ³⁶ It is interesting to note that a wide range of bio-compounds can take part in the biosynthesis of AuNPs, and these are abundant in plants. The chemical components of plants, such as flavonoids, phenols, quinones, and ketones, can reduce ions of precious metals, including Au, into NPs because these substances have many functional groups in their structure that can induce the stabilization and reduction of Au ions to AuNPs. ^{31 , 56} The hydroxyl groups present in polyphenols can be involved in the reduction of Au ions by their promotion to quinine forms, which follows Pearson’s Hard and Soft Acids and Bases (HSAB) principles. ³⁶ Based on these principles, soft acids prefer to coordinate with soft bases to produce covalent complexes, while hard bases choose to interact with hard acids to form ionic complexes. Indeed, the complex compound between a hard ligand and a soft metal (like Au⁺) is very unlikely to be formed. Flavonoids are another compound present in plants that play a significant role in the green synthesis of AuNPs. ³⁷ Flavonoids act as free radicals, transforming Au³⁺ ions to an intermediate complex, which is then oxidized to keto-form. The trivalent Au is finally reduced to AuNPs.

Using a green synthesis, palm (Elaeis guineensis) extract was used by Ahmad et al. ⁷⁵ to synthesize AuNPs with an average diameter of ∼ 28 nm. To remove the contaminations on the surfaces of the palm leaves, deionized water was used to clean them. After being sun-dried for a week, the leaves were air-cleaned to remove any dust that might have appeared on them during drying. They were then oven-dried for 8 h at 70 °C and crushed to acquire fine leaves powder. After drying, 5 g of the powder were added to 100 mL of water, and the resulting mixture was heated to 70 °C while stirring for 10 min. The mixture was then filtered through Whatman filter paper. To synthesize AuNPs, an aqueous solution of HAuCl₄ and palm leaf extract was combined in a beaker. FTIR spectra of the extract and colloidal solution revealed the formation of functional groups that can contribute to the reduction of Au ions to AuNPs. The most prominent changes were the shifting of the O–H stretching band from 3,435 cm⁻¹ to 3,450 cm⁻¹, confirming the presence of phenolic compounds. The shift of the stretching band associated with carboxylic and protein compounds at 1,634 cm⁻¹ to 1,620 cm⁻¹ was also demonstrated (Figure 7a). The majority of ofAuNPs had spherical shapes, with only a few AuNPs with triangular- and pentagonal shapes. In another study, Taib et al. ⁷⁶ utilized Hibiscus sabdariffa leaf extract as the reducing agent for the synthesis of AuNPs. After washing the leaves with water, they were dried at 65 °C for 48 h. The dried leaves were then ground into powder. An aqueous solution was made by dissolving approximately 1 g of the powder into 100 mL of distilled water. After removing the solid content through filtration, a light, orange-colored extract with 3.1 pH was obtained. An aliquot of 10 mL leaves extract, and 10 mL of distilled water were combined and stirred under normal temperature while adding 5 mL HAuCl₄·3H₂O solution. The transformation of the solution from a pale orange to a purple color indicated the formation of AuNPs. The calculation of NPs particle size using TEM revealed an average size of 7 nm when stirring took 80 min. The pattern of XRD was similar to the established XRD patterns for AuNPs, and the crystallite size was calculated to be 6.96 nm. The authors proposed that the caffeic acid moiety of the phenol-chlorogenic acid, which is abundantly present in the leaves extract, caused the reduction of Au³⁺ ions to Au⁰ (Figure 7b). The ortho-dihydroxyl groups of polyphenols that are converted to their oxidized form could stabilize the AuNPs.

Figure 7:

FTIR spectra of the AuNPs colloidal solution derived from Elaeis guineensis leaves extract, ⁷⁵ the possible reduction and stabilization mechanism of Au³⁺ ions to Au⁰ induced by caffeic acid moiety present in H. sabdariffa L. extract. Adapted from ref. 76].

3.3 Green synthesis of AuNPs using mushrooms

Mycomaterials are bio-compounds found in fungi like yeasts, molds, and mushrooms. The green synthesis of AuNPs from mycomaterials is referred to as myconanotechnology. ⁷⁷ Mushrooms are considered another versatile natural source for the synthesis of AuNPs due to their widespread availability in nature. ^{77 , 78} A mushroom is a macro fungus with main parts visible to the naked eye. ^{79 , 80} Only a small portion of 20,000 types of mushrooms have been thoroughly studied, and they are mostly unknown. Edible mushrooms such as Lentinus, Flammulina, Auricularia, and Pleurotus sp. have been discovered to show therapeutic properties, including anticancer, antioxidant, and antitumor activities.

The presence of diverse substances such as tannins, polysaccharides, phenolic compounds, and terpenoids in biomaterials is beneficial in the production of AuNPs. ⁸¹ The primary advantage of using mushrooms as a natural precursor for NP synthesis is due to the rich content of extracellular enzymes, which can act as a stabilizing agent for AuNP synthesis. Most of the NPs synthesized by edible mushrooms occur through the extracellular pathway, which takes place outside the mushroom cells, and only a small percentage of synthesis occurs intracellularly in the hyphae of mushrooms. ⁸² Generally, mushroom-derived AuNPs have spherical shapes and sizes ranging from 0.4 nm to about 300 nm; the majority of particles have sizes of <75 nm.

Dheyab et al. ⁸³ synthesized AuNPs using mushroom extract of Agaricus bisporus, which is commonly named Portobello mushroom. 20 g of freshly sliced Portobello mushrooms were used to make the watery extract. Mushrooms were first chopped and boiled in 150 mL of distilled water for 10 min. The crude solution was left to settle at 25 °C for a few minutes. Subsequently, the extract was purified using gauze and filter paper before being centrifuged for at 6,000 rpm 10 min 135 mL of HAuCl₄ was heated to 80 °C on a hot plate. Then, 15 mL of Portobello mushroom extract was mixed with the HAuCl₄ solution. After 30 min of stirring, the color of the mixture changed to pale magenta. When the mixture was further stirred for another 60 min, its color changed to a deep purple, and no further change was observed after. The FESEM exhibited the AuNPs with an average size of 53 nm and a variety of forms, including spherical, oval, triangular, drum-like, and hexagonal shapes. XRD patterns revealed the highest diffraction intensity at the (111) orientation, while UV–vis spectroscopy revealed the presence of an SPR peak at 568 nm. Another type of mushroom (Laetiporus versisporus), which is commonly called chicken of the woods, was used by Fathima et al. to synthesize AuNPs with a size of 10 nm. The fresh mushrooms were cleaned several times using water to remove the contaminants on the surface. The mushrooms are cut into small pieces and air-dried. The air-dried mushroom sample was then ground into powder. An aqueous solution of mushroom powder in distilled water was made, heated to boiling point, filtered, and cooled down to room temperature. The mushroom extract was mixed with HAuCl₄ solution. The mixture was left stirring in a dark place to achieve optimum reaction conditions. Stirring the mixture in this condition changed its original yellow color to a deep purple color. The purple AuNP product was centrifuged at 8,000 rpm for 10 min to separate the precipitate. The resulting precipitate was placed in hot air overnight to evaporate the water content, leading to the formation of AuNPs. The completeness of synthesis was done by UV–vis, which showed an absorbance peak of AuNPs at 566 nm, and TEM and SEM images, which clarified the spherical shape of AuNPs.

3.4 Green synthesis using algae

Algae are another novel source for the green synthesis of AuNPs. Algae are eukaryotic photoautotrophs that live in freshwater and oceanic habitats. ⁸⁴ Algae are also classified as unicellular microalgae (green algae, diatoms, and golden algae) and multicellular macroalgae (green algae, brown algae, and red algae) (Figure 8). Algae have tremendous potential for the synthesis of AuNPs due to the existence of unique chemicals such as neutral glucan, fucoidan, and alginic acid that are absent in other natural organisms. ³⁷ Algae are the fastest-growing organisms that are readily available in watery environments. Thus, a great advantage of using algae biomass as the reducing agent for the synthesis of AuNPs is their low-cost cultivation from water. ⁶⁹ Algae offer abundant heavy metal binding sites, which facilitate metal absorption due to the existence of many hydroxyl groups in their subcomponents, such as proteins and polysaccharides (sulfated fucoidans). ^{37 , 69}

Figure 8:

Classification of algae into macroalgae and microalgae based on photosynthetic pigments, illustrating diverse biological sources for green synthesis of AuNPs. Adapted from ref. 85].

Senthilkumar et al. ⁸⁶ used a red marine macroalgae (Gelidiella acerosa) to synthesize AuNPs. The algae were subsequently cleaned with clean water and deionized water to eliminate any contaminants and remaining salted materials on the surfaces, respectively. The cleaned algae were allowed to air-dry at room temperature for 2 weeks slowly. The dried algae were ground to powder using a household mixer. An aqueous solution was made by adding 5 g of algal powder to 100 mL of boiling distilled water. The resultant mixture underwent continuous stirring at 60 °C for 20 min. A Whatman filter paper was used to filter the green-colored watery extract. To synthesize AuNPs, 10 mL of the filtered extract was mixed with 90 mL of HAuCl₄ solution. Following that, the solution was maintained at 37 °C in a static condition. After 1 h of stirring with exposure to sunlight, the light-yellow color of HAuCl₄ solution changed to ruby red, indicating the generation of AuNPs. The sizes of particles ranged from 5.81 to 117.59 nm as confirmed from the high-resolution TEM. UV–vis spectroscopy analysis demonstrated the SPR peak at 526 nm (Figure 9). After a 3-month period, the UV–vis was retaken, yielding an almost identical peak at 526 nm, indicating the high stability of the solution over time. However, the intensity of this peak steadily grew, providing confirmation of the ongoing growth and development of the AuNPs. The authors proposed that the reasons behind the high stability of AuNPs could be due to the secondary metabolites present in the algae. The SEM investigation visualized the shapes of AuNPs in spherical, cubical, and hexagonal forms. In another research, AuNPs with an average diameter of 5 nm were synthesized by Suganya et al. ⁸⁷ using blue-green algae (Spirulina platensis). 1 g of the powdered algae was combined with 10 mL of distilled water. After extraction and drying, the mixture was centrifuged at 5,000 rpm for 10 min to obtain the final extract. An aliquot of 10 mL extract was mixed with 10 mL of HAuCl₄ solution to initiate the AuNPs synthesis. Sodium hydroxide (NaOH) was then added to the extract/HAuCl₄ mixture while stirring. The addition of NaOH caused an immediate color shift from green to greenish-yellow. The stirring was continued at room temperature for 3 h before incubating for 48 h. The generation of AuNPs was visually confirmed by the appearance of a ruby-red color mixture. High-resolution TEM showed the monodispersed AuNPs with spherical shapes. FTIR and Raman analyses described the involvement of carboxylate, hydroxyl, and amine functional groups in the reduction and stabilization of AuNPs.

Figure 9:

UV–vis analysis of AuNPs derived from Gelidiella acerosa, showing stability over a 3-month period, indicating the potential of algae-derived AuNPs for biomedical applications. Adapted from ref. 86].

3.5 Green synthesis using yeasts

Yeasts are unicellular eukaryotic microfungi that can be found almost anywhere in any environment. ⁸⁸ Indeed, they can be found in water, soil, fruits, skin surfaces, and even in the gastrointestinal tract of warm-blooded animals. These heterotrophic microorganisms can get their energy and nutrients from both living and dead residual organic matter. This high production of bio-compounds with carboxyl and amine groups, glycans, and sugars with reduction capability make yeasts a promising candidate for synthesizing AuNPs. In a similar mechanism to algae, yeasts can efficiently absorb and accumulate a wide range of heavy metal ions from their surroundings. ⁸⁹ The reasonably simple nutrients needed for the rapid growth of yeasts and synthesis of enzymes, as well as the ease of culturing under laboratory conditions, made them preferable to bacteria for synthesizing AuNPs. ⁹⁰

Zhang et al. ⁹¹ used an isolated yeast from sea mud (Magnusiomyces ingens) to synthesize AuNPs, which had a size of ∼80 nm. After incubation, the cells were centrifuged for 10 min at 10,000 g at 4 °C, washed two times with distilled water, and kept at −80 °C. To synthesize AuNPs, the yeast cells were subsequently resuspended in the water with a ratio of 2.2 and mixed with HAuCl₄ solution. The mixture was left stirring at 30 °C overnight. The mixture was then centrifuged at 3,000 g for 5 min to obtain the supernatant, which was filtered through a 0.45 µm syringe Millipore filter to eliminate cell fragments. UV–vis revealed a narrow absorbance peak that remained unchanged during reaction time, suggesting the existence of capping agents released by the yeast cells that prevent AuNPs agglomeration. The SEM and TEM studies showed diverse irregular morphologies, including sphere and plate-like (hexagon, triangle, pentagon) (Figure 10). Using a similar pathway, Rónavári et al. ⁹² used Phaffia rhodozyma, a kind of red yeast, to synthesize spherical AuNPs with extremely small size of 2.22 nm. The yeast cells were harvested after incubation for 5 days. 10 g of fresh biomass was immersed in 200 mL of sterile water. The size of cells in water was broken down in a bead beater using glass beads. The small-sized cells were centrifuged for 15 min at 18,000 g at 4 °C to eliminate large cell fragments. The resulting product was then filtered through using a cellulose nitrate filter with pores of 0.45 µm. A similar protocol was used for synthesizing AuNPs. 10 mL of HAuCl₄ solution was added to 90 mL of extract to make a suspension. The suspension was continuously stirred at 22 °C overnight which led to the formation of NPs. The AuNPs were centrifuged at 18,000 g for 15 min at 4 °C and washed twice with distilled water. The synthesized spherical AuNPs were essentially monodispersed and well separated. The existence of SPR peak was confirmed at 541 nm, and the XRD analyses confirmed the crystalline patterns of AuNPs (Table 2).

Figure 10:

The SEM (a) and TEM (b) images of synthesized AuNPs by Magnusiomyces ingens LH-F1 yeasts. Adapted from ref. 91].

5.1 Antibacterial activities

Recent advancements in medicine have led to the development of new drugs for bacterial infections, yet the overuse of multiple drugs has contributed to the rise of drug-resistant microorganisms, a serious public health concern. ¹⁹ This emphasizes the need to explore novel approaches, such as AuNPs, which offer potentially modifiable antibacterial properties. Green-synthesized AuNPs have shown promising antibacterial effects, potentially due to natural bio-compounds present in plant or algae extracts that may enhance bactericidal action. ⁹³ However, the antibacterial efficacy of AuNPs is influenced by various factors, including synthesis methods, AuNP concentrations, and bacterial strains. ⁹⁴ Although positively charged AuNPs generally show high affinity for negatively charged bacterial cell surfaces, the degree of susceptibility varies between Gram-positive and Gram-negative bacteria due to differences in their cell wall structures. ³⁷ Although all bacterial cells are negatively charged and have a high affinity to interact with positively charged AuNPs, Gram-negative bacteria are more vulnerable to AuNPs due to their thinner peptidoglycan layer than Gram-positive bacteria. Antibacterial action initially happens when AuNPs tightly attach to the bacteria’s surface. This will lead to the destruction of flagella, visible damage to cell membranes, and reduced biofilm formation. ⁹⁵

For example, Suganya et al. ⁸⁷ synthesized 5-nm spherical AuNPs using the blue-green algae (S. platensis) and showed their effective antibacterial activities against two Grain-positive bacteria, Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis). To assess the antibacterial activity, the zone of inhibition for each bacterium was measured (Figure 11), and vancomycin was used as the positive control. The antibacterial activity noticeably increased when the concentration of AuNPs increased. The colony-forming unit (CFU) method was also used to evaluate the bactericidal capacity of AuNPs. The results showed the maximum reduction capability of 80.5 % and 97.4 % for S. aureus and B. subtilis colonies when treated with AuNPs of 150 μg/mL, respectively. However, as the authors expected, when colonies were exposed to higher concentrations of 200 μg/mL, a nearly complete reduction of 99 % was observed. Another representation of this concept was shown by Sunderam et al. ⁹⁶ demonstrated that AuNPs synthesized with Anacardium occidentale leaves extract were effective against Gram-positive B. subtilis and Gram-negative E. coli, showing higher activity against E. coli. The differences in inhibition zones (8 and 10 mm for B. subtilis, and 18 and 24 mm for E. coli) reflect variations in bacterial susceptibility and suggest that further investigations are needed to understand the mechanisms driving these differences.

Figure 11:

Zone of inhibition diameters for B. subtilis and S. aureus after exposure to varying AuNP concentrations, illustrating antibacterial efficacy. Adapted from ref. 87].

5.2 Anticancer activities

Cancer is a leading cause of death in humans. The number of different cancer cases has risen to an alarming level during the past years. ³⁷ While nanotechnological breakthroughs have paved the way for countless methods for cancer diagnosis and therapy, most of these techniques showed potential side effects or drug reactions. As such, there is a tremendous potential to develop innovative drugs with minimal toxicity or side effects in a sustainable manner. Given the outstanding capacity of bio-compounds to readily adhere to the surfaces of AuNPs, the use of green synthesized AuNPs for treating tumors has been recommended. ⁹⁷ This asset sets green synthesis apart from traditional ones in terms of its role in fighting against cancer. The remarkable ability of green synthesized AuNPs to permeate into tumor-bearing tissues is a great advantage that increases their likelihood of being utilized in cancer treatment. ⁹⁸ It goes without saying that smaller AuNPs have stronger anticancer activities and greater renal clearance than large AuNPs. The AuNPs with relatively small sizes demonstrated better penetration into tumor tissue. ⁹⁹ Using capping agents can unquestionably contribute to synthesizing AuNPs with robust antiproliferative activities that can disrupt cancer cell proteins or enzymatic activity. ³⁷ The accumulation of AuNPs in cancer cells and the generation of reactive oxygen species (ROS) could induce cell apoptosis via intracellular oxidative stress. ^{100 , 101} For instance, AuNPs can increase the levels of ROS production in HeLa (human cervical cancer) cells and cause apoptotic cellular death through the mitochondrial-mediated pathway. ¹⁰⁰

Wu et al. ¹⁰² synthesized spherical AuNPs with a size of 200 nm from Siberian ginseng (Eleutherococcus senticosus). The authors used different concentrations of AuNPs (0.5, 1, 2.5, 5, 10, and 20 μg/mL) to treat B16 murine melanoma cells both in vitro and in vivo. The treatment with AuNPs significantly decreased the number of viable cells. The reduction in the cell viability occurred in a dose-dependent fashion after exposure to a dosage of 0.5–20 μg/mL, with a 50 % reduction observed at the dose of 10 μg/ml AuNPs, as seen in Figure 12. Clarance et al. ¹⁰³ used AuNPs synthesized from endophytic fungi (Fusarium solani) to evaluate their anticancer activities against HeLa cervical cancer cells, MCF-7 human breast cancer, and HEK human embryonic kidney cells. Cells were exposed to different concentrations of spindle-shaped AuNPs (0.5–4 μg/mL). The findings showed that AuNPs delivered anticancer properties against MCF-7 and HeLa cancer cell lines with IC₅₀ values (half-maximal inhibitory concentration) of 0.8 μg/mL and 1.3 μg/mL, respectively. However, the findings showed negligible anticancer activities against HEK cancer cell lines. The irregular morphology of MCF-7 cell lines has likely resulted from the apoptotic fragmentation of cells. Hosny et al. ¹⁰⁴ designed a study to compare the anticancer activities of spherical AuNPs synthesized using Atriplex halimus (2–10 nm) and Chenopodium amperosidies (up to 40 nm). AuNPs synthesized using A. halimus exhibited higher efficiency in reducing the growth of MCF-7 cell lines than those synthesized using C. amperosidies. The recorded cell viability IC₅₀ values were 47.03 and 22 μg/mL for A. halimus-AuNPs and C. amperosidies-AuNPs, respectively.

Figure 12:

Anticancer activities of the green-synthesized AuNPs from Siberian ginseng against B16 cancer cells. Adapted from ref. 102].

5.3 Cancer imaging and diagnostic applications

The early diagnosis and detection of cancer is critical to reduce cancer mortality. Using AuNPs for cancer imaging is highly beneficial due to their distinctive capabilities such as light absorption and scattering characteristics. ¹⁷ Raman imaging using non-emitting near-infrared wavelength is a common tool to visualize tumor-bearing tissues and track tumor progression. Using AuNPs in Raman imaging is very useful because the amplitude of the Raman signals on the AuNP surface is exceptionally high. ¹⁰⁵ The high Raman signals of AuNPs stem from the presence of a broad SPR peak and the fast transfer of charges between the AuNP’s surface and the target cancer cells. To enable better diagnosis or treatment follow-up and to obtain a more detailed image of tumors, computed tomography (CT) imaging is performed by intravenous injection of Iodinated contrast agents. ¹⁷ However, AuNPs have proven to be generally better than iodine-based contrast agents in terms of giving better contrast and imaging windows. ¹⁰⁶ Other imaging procedures, such as positron emission tomography (PET) and magnetic resonance imaging (MRI), can also benefit from the development of AuNPs as targeted contrast agents. The site-specific accumulation of AuNPs in cancer cells can enable their early detection without side effects.

Morel et al. ¹⁰⁷ used Hypericum lanceolatum (H. lanceolatum) as a traditional medicinal plant to synthesize flower-shaped AuNPs (40–80 nm). The potential application of plant-mediated AuNPs in visualizing cancer cells using CT-scan imaging was investigated. The author observed that H. lanceolatum-extract and citrate extract did not yield sufficient contrast to be visible on tumor images. In contrast, H. lanceolatum-derived AuNPs exhibited a strong contrast, as shown by the presence of a minor black deposit on the tube’s base. The radiological contrast of the deposit was measured at around 621 HU, whereas that of the supernatant only showed 25 HU. Given these remarkable imaging features, the authors proposed that AuNPs could have promising potential to be used as photoacoustic contrast agents for multiple imaging modalities such as CT scans and MRI. Another study conducted by Uthaman et al. ¹⁰⁸ used mannan (a bioactive polysaccharide derived from the cell walls of baker’s yeast Saccharomyces cerevisiae) to synthesize spherical AuNPs with an average size of 9.18 nm. The authors first carried out CT phantom tests to examine the possible applications of AuNPs as a contrast agent for CT imaging, which showed a CT signal of 303 HU measured for AuNPs with a concentration of 20 mg/mL. Subcutaneous injection of AuNPs was inoculated into C57Bl/6 mice at the hindlimb footpads to understand the in vivo viability of AuNPs for lymph node CT imaging. It was found that injection of mannan-synthesized AuNPs into popliteal lymph nodes showed better contrast visibility than sodium citrate-synthesized AuNPs (Figure 13). The visible contrast of AuNPs on lymph nodes showed a time-dependent manner and significantly increased from 86 to 618 HU during 20 h of imaging. This observation clearly confirmed the effective diffusion of AuNPs from the injection site throughout the lymphatic system over time.

Figure 13:

In vivo CT images of popliteal lymph nodes of mice that were injected with citrate-synthesized AuNPs (a) and mannan-synthesized AuNPs (b) over 20 h imaging period. Adapted from ref. 108].

5.4 Targeted drug delivery and cancer therapy applications

AuNPs have contributed to modern medicine not only for cancer diagnostic applications but also for their ability to load high dosages of therapeutic agents and unload at the target cancer sites with enhanced therapeutic efficiency and much less adverse reactions. ¹⁸ The ability to customize AuNPs offers many opportunities for the creation of drug nanocarriers with varying diameters, shapes, and size distributions. ^{17 , 109} The large surface-area-to-volume ratio of AuNPs enables the efficient absorption of therapeutic agents onto their surface. ¹⁷ There are two methods to bind therapeutic agents onto the surface of AuNPs: (i) via linker molecules (also known as conjugation) and (ii) non-covalent bonds (also known as sorption). The versatility and controlled release of AuNP coatings with diverse therapeutic agents make them a promising nanoplatform for cancer treatment. ¹¹⁰

Laksee et al. ¹¹¹ synthesized spherical AuNPs with a diameter of 13.7 nm from pullulan. The potential of AuNPs, which were conjugated with pullulan moieties (PM), was investigated for the targeted delivery of doxorubicin (DOX) as an anticancer drug without side effects. DOX-loaded AuNPs@PM was demonstrated to induce more apoptosis and higher anticancer activities against lung tumor cells than free DOX while exhibiting negligible cytotoxicity against normal lung cells. Malathi et al. ¹¹² used chitosan to produce spherical AuNPs with sizes of 2–3 nm. They were then used as nanocarriers for the controlled release of an antibiotic, rifampicin (RIF), which is often used to treat tuberculosis. The drug loading efficiency of RIF-loaded AuNPs was calculated to be 71 %. The efficacy of RIF-loaded AuNPs exceeded that of free RIF in terms of antibacterial effectiveness because RIF-loaded AuNPs could better penetrate the cell walls of bacteria than free RIF. RIF-loaded AuNPs were reported to confer antibacterial action against both the Gram-negative (Pseudomonas aeruginosa) and Gram-positive (B. subtilis) bacteria.

5.5 Current clinical trials involving AuNPs

AuNPs have shown considerable promise in a variety of clinical applications due to their unique physicochemical properties and biocompatibility. In molecular imaging, AuNPs serve as effective contrast agents, particularly in photoacoustic imaging. With their high absorption cross-sections, AuNPs greater than 20 nm have proven effective in enhancing imaging contrast in in vivo models, although non-biodegradable properties can pose challenges, such as potential accumulation in organs like the spleen and liver. ^{113 , 114} However, recent studies have indicated that smaller, sub-10 nm AuNPs, which are comparable in size to biomolecules such as albumins and antibodies, may offer a solution to these issues by achieving highly uniform tissue distribution. In murine breast cancer models, these ultrasmall AuNPs demonstrated effective tissue penetration and distribution, positioning them as a promising option for sensitive and targeted in vivo imaging of cancer cells. ¹¹⁵

In the realm of medical implants, AuNPs are being integrated to enhance osseointegration and reduce bacterial growth. For example, titanium implants coated with AuNPs (Ti-GNPs) have been shown to significantly promote osteogenic differentiation by upregulating mRNA expression specific to osteogenesis in human adipose-derived stem cells (ADSCs). ¹¹⁶ Additionally, studies using Ti-GNPs in animal models revealed improved osseous interface formation without signs of cytotoxicity, presenting AuNPs as an attractive option for enhancing implant performance and longevity. Further research on AuNP-mediated gene delivery is underway, focusing on its potential to improve bone formation in dental implants, even under conditions of osteoporosis. ^{117 , 118}

AuNPs have also been widely studied in diagnostics and biosensing. Their unique properties make them ideal for electrochemical immunosensors, as evidenced by clinical investigations for detecting disease markers such as Hepatitis B surface antigens and diphtheria toxins. In particular, AuNP-based platforms have demonstrated the ability to amplify the electrochemical signal of antibody-antigen interactions, enhancing the sensitivity and specificity of these diagnostic tools. ¹¹⁹ Paper-based devices incorporating AuNPs are also gaining attention for rapid, portable biomarker detection, with successful applications for identifying biomarkers of diseases like tuberculosis and infectious eye diseases using mechanisms like surface-enhanced Raman scattering (SERS) and colorimetric detection. ^{120 , 121}

In radiopharmaceuticals and nuclear medicine, AuNPs conjugated with radioisotopes such as Technetium-99m (Tc-99m) have been explored for both diagnostic and therapeutic uses, a field known as theranostics. One study demonstrated that Tc-99m-labeled AuNPs targeting atherosclerotic plaques enabled improved imaging diagnosis via SPECT/CT, with AuNPs showing enhanced blood circulation times and strong affinity to targeted sites. ¹²² Another investigation into Au nanoclusters containing radioactive isotopes illustrated their effectiveness in eradicating various cancer cell lines, such as melanoma and breast cancer, while also serving as PET imaging agents. These dual-function AuNPs radiopharmaceuticals are paving the way for simultaneous imaging and therapy applications in cancer treatment. ^{123 , 124}

Finally, AuNPs are proving invaluable in lab-on-a-chip technologies, where they are used in developing compact, efficient diagnostic devices. For instance, a recent clinical study integrated AuNPs into a lab-in-a-syringe system designed for immunosensing biomarkers, successfully detecting human IgG and PSA levels in spiked urine samples with high sensitivity. ¹²⁵ Other lab-on-a-chip platforms utilizing AuNPs have enabled advancements in detecting environmental pollutants and biological markers, which may extend to critical healthcare applications such as viral and bacterial pathogen detection. ¹²⁶

These ongoing trials underscore the potential of AuNPs to enhance the sensitivity, specificity, and multifunctionality of diagnostic and therapeutic applications, marking significant steps toward clinical integration across diverse medical fields.