Size-Dependent Antioxidant Effects of Gold Nanocrystalline Films

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Suliasih, Anis Sakinah, Marissa Angelina, Haliza Katas, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7233568/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Dec, 2025 Read the published version in BioNanoScience → Version 1 posted 11 You are reading this latest preprint version Abstract The exceptional chemical and physical properties of gold nanoparticles (AuNPs) have led to their significant use in both medicinal and cosmetic applications. This study investigated the antioxidant properties of AuNPs. Various sizes of AuNPs were synthesized through the electrodeposition technique, resulting in the formation of deposited films. Structural analysis with an X-ray diffractometer (XRD) revealed that a face-centered cubic structure of metallic gold with fine crystallite size (19 to 31 nm) was successfully formed on the substrate. Morphological studies employing a field emission scanning electron microscope (FESEM) showed that the deposited film consisted of relatively fine particles, providing numerous active sites for the radical scavenging mechanism. Antioxidant assays demonstrated that the deposited film exhibited promising antioxidant activity, particularly with a large number of very fine gold particles. This efficacy was evidenced by their efficacy in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, with a maximum percentage inhibition of 43.91%, and the Ferric Reducing Antioxidant Power (FRAP) assay, with a maximum percentage reduction of 38.22%. These results highlight the notable antioxidative capabilities of AuNPs, especially those produced under specific experimental conditions. This suggests their potential in tackling problems related to oxidative stress. gold nanoparticle antioxidant electrodeposition film particle size Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Free radicals are molecular species that possess at least one unpaired electron in their outer shell. These species can arise as a result of the oxidative phosphorylation respiratory cycle, which can interact with biological macromolecules including cellular DNA, leading to single-strand and double-strand breaks that ultimately can initiate cellular aging, mutagenic alterations, cardiovascular disorders, and the development of tumors or cancer (Jomova et al . 2023). Oxidative stress refers to the physiological state that occurs when an organism’s internal defence mechanisms, encompassing enzymatic, non-enzymatic, and dietary components, are inadequate to counteract an excessive buildup of free radical species. This condition leads to the oxidative damage to both extracellular and cellular macromolecules, such as lipids, proteins, and nucleic acids, ultimately resulting in tissue injury (Chen et al . 2024). However, in general, these free radical reactions can be inhibited or mitigated by certain antioxidant compounds obtained from natural or synthetic sources [ 3 ]. Antioxidants are defined as compounds that possess the ability to delay, regulate, or inhibit the process of autoxidation (Chukiatsiri et al . 2024). They play a crucial role in human health by impeding or delaying undesired oxidation reactions, thereby preventing oxidative stress associated with diseases such as hypertension, neurodegenerative disorders, or cancers [ 1 ]. Both natural and synthetic antioxidants have limited effectiveness due to poor absorption, difficulty in entering cells, and breakdown during delivery. However, the current progress in nanotechnology has facilitated the development of a variety of inorganic nanoparticles that exhibit enhanced antioxidant properties, thus offering promising potential for various applications (Khalil et al . 2020, [ 5 , 6 ] One of the nanomaterials that can function as an antioxidant is gold nanoparticles (AuNPs). AuNPs have been extensively employed in the recent research due to their unique chemical and physical properties (Dong et al . 2024). Studies show that AuNPs act as antioxidants, demonstrating strong kinetic effects in scavenging of reactive oxygen species (ROS) within living cells [ 4 ]. The kinetic effect is greatly affected by the size of AuNPs. Smaller particles have been shown to enhance catalytic activity due to their ability to generate a larger surface area, facilitating a greater number of active sites available for interaction with free radicals (A. Bano et al . 2023; Suliasih et al ., 2024). Moreover, numerous studies have shown that AuNPs possess a notable antioxidant effect, highlighting their potential in medical applications including wound healing, anti-asthma, antipyretic, anti-cancer and antidiabetic agents. Additionally, the relatively low toxicity of AuNPs in human subjects increases their desirability for use in therapies [ 7 , 11 – 14 ]. In antioxidant research, AuNPs are primarily synthesized through biosynthesis methods [ 11 , 15 – 17 ]. This approach mainly utilizes bioactive phytochemical compounds with antioxidant properties to reduce gold ions and form AuNPs. Since the antioxidant activity exhibited by this AuNPs do not originate solely from the AuNPs themselves, this study aimed to determine the antioxidant activity response of AuNPs themselves [ 8 , 18 , 19 ]. For this purpose, AuNPs were produced as a solid phase or thin film without employing any antioxidant compounds in their synthesis using electrodeposition method. Electrodeposition is known as a facile and low-cost method to directly grow metallic deposit in a form of uniform films [ 20 , 21 ]. This allows the observation of the mechanism of single AuNPs as antioxidants. This study is aiming to determine the effects of gold particle size on their antioxidant activities by varying the scanning rate of the electrodeposition process. Materials and Method The materials used in this study were gold(III) chloride trihydrate (HAuCl 4 ·3H 2 O), sulphuric acid (H 2 SO 4 ), 2,2-diphenyl-1-picrylhydrazyl (DPPH) ethanol, sodium acetate trihydrate, distilled water, TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), hydrochloric acid, ferric chloride, trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), Fluorine doped Tin Oxide (FTO) glass as the substrate, and 24-well microplates as the sample containers for testing. Synthesis of AuNPs The synthesis of AuNPs was carried out in the electrolyte solution containing 0.5 mM gold solution (HAuCl 4 ·3H 2 O) dissolved in 0.5 M H 2 SO 4 . By employing the cyclic voltammetry method, AuNPs were grown with a starting voltage of -1.0 V vs Ag/AgCl and a final voltage of 1.5 V vs Ag/AgCl. The scan rate variations employed in the study were 375, 300, 250, 125, and 80 mV/s. This electrochemical deposition was performed in a three-electrode cell with Ag/AgCl acted as the reference electrode, FTO served as the working electrode, and a platinum plate was functioned as the counter electrode. The experimental procedures were conducted at 25°C. Once the synthesis was finished, the deposited AuNPs on the substrate were subjected to rinsing by distilled water, and subsequently dried. Characterization The structural analysis of the electrodeposited AuNPs was conducted using a SMARTLAB Rigaku X-ray diffractometer (XRD) with Cu-K-Alpha1 radiation (λ = 1.540598 Å). A Scanning Electron Microscope (Thermo Scientific: Quanta 650) and an Energy Dispersive X-Ray Spectrometer (Qxford Instrument: Xplore 15) were employed to investigate the morphological and elemental analysis, respectively. The particle size distribution of the deposited AuNPs was determined from the obtained SEM micrographs using ImageJ software. Antioxidant determination The antioxidant activity response of AuNPs was evaluated using in vitro test methods specifically the DPPH and Ferric Reducing Antioxidant Power (FRAP). DPPH Assay The absorbance of samples was measured by a Spectrophotometer (Microplate Reader Thermo Scientific: Elisa Reader). Deposited AuNPs (dimension of 5 mm x 15 mm) were distributed into a 24-well microplate and subsequently submerged in 1.3 ml of 100 µM DPPH-ethanol solution. Afterward, the samples were subjected to agitation through shaking and the absorbance was measured using a spectrophotometer at 300–750 nm at the pre-determined time intervals: 15, 30, 45, 60, 75, 90, 105, 120, 135, and 150 minutes. The percentage inhibition of DPPH was determined using Eq. ( 1 ). $$\:\%Inhibition=\frac{Control\:Absorbance-Sample\:Absorbance\:}{Control\:Absorbance\:}\:x\:100\%$$ 1 The control absorbance is the peak absorbance exhibited by the DPPH solution, and the sample absorbance is the peak absorbance seen in the DPPH solution after adding AuNPs samples at a wavelength of 518 nm. FRAP The sample was prepared similarly to the DPPH test. The FRAP solution consisted of sodium acetate trihydrate (0.3 M; pH 3.6) + TPTZ (2,4,6-tris(2-pyridyl)-s-triazine (10 mM/L)) in 40 mM HCl + ferric chloride (20 mM) with a ratio of 10:1:1, and then its absorbance was measured at a wavelength of 596 nm. To measure the antioxidant activity of AuNPs in reducing Fe3+, the Eq. (2) was used. $$\:\%Reduction=\frac{Sample\:Absorbance-Control\:Absorbance\:}{Sample\:Absorbance\:}x\:100\%$$ The sample absorbance refers to the absorbance value of the FRAP solution after the addition of AuNPs samples, while the control absorbance represents the maximum absorbance value of the FRAP solution. Results and Discussion Characterization XRD The XRD results of the AuNPs electrodeposited using cyclic voltammetry technique with different scan rate are shown in Fig. 1 a. The peaks of Au crystal appeared at 2θ 38.32°, 44.45°, 64.68°, and 77.72° that could be associated to the reflection from (111), (020), (022) and (131) planes, respectively. This pattern is agreed with the crystal open database (COD) No. 96-901-1614 for metallic Au phase. Though, the peaks at 2θ 38.32° was observed to be overlap with the peak of fluor tin oxide substrate at 2θ 37.87°. However, the magnified patterns, which are shown in Fig. 1 b, indicate the peaks broadening and shifting to higher 2θ clearly confirm that the reflection from (111) plane of Au was observed. In this case, the high peaks of the substrate were due to the very thin Au films that allow high intensity reflection from the substrate. The crystallite size of Au calculated by following Scherrer Eq. ( 3 ) indicates that the fine crystallites ranging from 19 to 31 nm were successfully grown by this approach. $$\:D=\frac{K{\lambda\:}}{\beta\:\text{cos}\theta\:}$$ 3 Where D is crystallite size, K is shape factor, λ is the X-ray radiation wavelength, β is peak broadening, and θ is the Bragg angle. EDX & SEM The characterization results using EDX confirm the existence of Au element on the layer of the substrate. Figure 2 presents the spectrum signals of AuNPs ranging from 2.1 to 12.7 keV. In addition to the presence of Au, spectra corresponding to other elements from the FTO substrate are also observed, namely Sn and Si. The results of SEM for AuNPs synthesized with varying scan rates are depicted in Fig. 3 . The resulting particle morphology ranges from spherical to irregular, with some particles overlapping and merging to form large nanoclusters. As the scan rate was gradually increased, the particle size appeared to be decreased while the number of particles appeared to be increased. Subsequently, the number of nanoclusters decreased, resulted in well dispersed nanoparticles. The SEM images were then processed to ImageJ software for determining its particle size as depicted in Table 1 . The particle size results indicate that the faster scan rates produced finer particles compared to that of slower scan rates. This phenomenon arises because increasing the scan rate shortens the particle growth phase, leading to the formation of smaller nanoparticles [ 22 ]. Conversely, slower scan rates yield larger AuNPs due to the prolonged particle growth, which increases the likelihood of particle overlap, resulting in agglomeration and subsequent enlargement of particles. Table 1 Particle size resulting from variations in scan rate Scan Rate (mV/s) Particle Size (nm) 80 88.96 125 78.16 250 71.55 300 66.8 375 51.75 Antioxidant activity test DPPH This DPPH assay is the simplest and most widely used method for determining antioxidant activity. It involves the conversion of free radical into non-radical species through the presence of antioxidant compounds [ 23 ]. Figure 4 illustrates a decrease in DPPH absorbance values across all AuNPs samples. The data clearly show a consistent decline in absorbance over the incubation period for each sample. This trend signifies the ability of AuNPs to inhibit DPPH. The level of DPPH inhibition was quantified as a percentage, providing a metric for evaluating the antioxidant efficacy of AuNPs. The data in Fig. 5 indicates that the sample prepared at the scan rate of 300 mV/s exhibits the highest inhibition activity of 43.91%. This result correlates with the particle size, as antioxidant activity increases with higher scan rates. As previously mentioned, the particle size obtained at this scan rate was 66.8 nm, which is lower than that achieved at lower scan rates (80 mV/s to 250 mV/s). These smaller particles result in a large of surface area, contributing to increased antioxidant activity. However, an anomaly is observed for the sample produced at 375 mV/s, where the inhibition is lower than that of the 300 mV/s sample but still higher than the samples produced at 250 mV/s, 125 mV/s and 80 mV/s. This phenomenon may be attributed to the reduced number of particles formed on the substrate, as illustrated by the SEM micrograph (Fig. 3 e). These findings indicate that the optimal active sites for reaction with DPPH were achieved at a scan rate of 300 mV/s. AuNPs can donate electrons to unpaired DPPH radicals, reducing DPPH to form stable non-radical compounds [ 24 ]. The proposed quenching mechanism involves electron transfer from AuNPs to stabilize the nitrogen atoms in DPPH by forming coordinated covalent bonds. This observation leads to the formation of a coordination covalent bond between gold and nitrogen (Au-N), which effectively quenches the initially radical DPPH compound. The stabilization of the nitrogen atoms by the gold atoms is a significant factor in this process [ 25 ]. The quenching process of free DPPH radicals is characterized by a colorimetric shift from purple to yellow, which signifies the conversion of free DPPH radicals into stable compounds. Figure 6 shows an illustration of the assumed mechanism of quenching free DPPH radicals by AuNPs. FRAP The FRAP method is used to validate the findings of AuNPs samples identified by the DPPH method. Table 2 shows that the sample prepared at a scan rate of 300 mV/s exhibited the highest reduction percentage, reaching 38.22%. The findings indicate a notable rise in absorbance in the FRAP samples that immersed with AuNPs/FTO. Table 2 The correlation between the scan rate and the percentage of inhibition in the FRAP test Sample Reduction (%) 80 mV/s 16,10 125 mV/s 16,14 250 mV/s 17,88 300 mV/s 38,22 375 mV/s 35,00 This phenomenon may demonstrate a reduction reaction of Fe 3+ complex compounds to Fe 2+ complex compounds as a result of electron donation from AuNPs [ 26 ]. This observation provides evidence for the presence of antioxidant activity. The findings are consistent with the analysis carried out utilizing the DPPH method. When correlated with SEM data, it is suggested that the sample scanned at a rate of 300 mV/s showed the highest levels of inhibition and reduction, due to its abundance of spherical to irregular-shaped particles. It is hypothesized that the irregular-shaped particles contain many active sites, edges, or corners, leading to increased conductivity [ 27 ] and displaying greater surface reactivity [ 28 ], ultimately enhancing their effectiveness as antioxidants. Figure 7 depicts the correlation between scan rate and reduction percentage in the FRAP test, as well as inhibition percentage in the DPPH test. The findings in Fig. 7 indicate that the reduction percentage obtained via the FRAP method is comparatively lower than that observed through the DPPH method. The suggests that the AuNPs synthesized using the FRAP method may have suboptimal properties for reducing FeIII-TPTZ complexes under thermodynamic conditions, resulting in slower reaction rates [ 29 ]. However, these AuNPs are highly effective at quenching DPPH radicals, leading to the highest inhibition values observed with the DPPH method. Conclusion AuNPs synthesized through the electrodeposition method at different scan rates, demonstrate considerable antioxidant potential. This is evidenced by their capacity to interact with DPPH radicals and to reduce Fe 3+ ions effectively. Specifically, AuNPs synthesized at a scan rate of 300 mV/s were shown to inhibit DPPH free radicals by 43.91% and reduce Fe 3+ ions by 38.22%. The observed increase in antioxidant activity is likely attributable to the reduced particle size of the AuNPs. This reduction in size increases the available surface area and the density of the active sites, thereby promoting more efficient radical scavenging and ion reduction processes. These findings underscore the remarkable antioxidative properties of AuNPs, particularly those synthesized under specific conditions, highlighting their promising role in combating oxidative stress-related disorders. The irregular morphology of these AuNPs enhances their surface area, facilitating greater interaction with reactive species and enhancing their antioxidant efficacy. Furthermore, the optimal synthesis conditions, as demonstrated by the selected scan rate, highlight the critical significance of meticulous control over experimental parameters in the customization of the properties of AuNPs for targeted applications. These results pave the way for further exploration and utilization of AuNPs as potent antioxidants in various biomedical applications. Declarations Competing interests. The authors declare no competing interests. Ethics Declaration. Not Applicable. Author Contribution B.A.S.: Conceptualisation, Methodology, Investigation, Data Analysis, Writing_original draft, Writing_review & editing; A.S: Investigation, Data Analysis, Project Administration; M.A.: Resources, Supervision; H.K.: Conceptualisation, Methodology, Resources, Supervision, Writing_Review & Editing, Funding Acquisition; S.B: Conceptualisation, Methodology, Resources, Funding Acquisition, Supervision, Writing_Review & Editing, All authors have read and approved the final manuscript Acknowledgement The authors acknowledge the financial support provided by the Lembaga Penelitian dan Pengabdian Masyarakat (LPPM) at Universitas Negeri Jakarta through the International Collaborative Research scheme, under contract number: 19/KI/LPPM/III/2025, and Universiti Kebangsaan Malaysia through a research grant (Geran Universiti Penyelidikan UKM (GUP-2022-004) Data availability No datasets were generated or analysed in the course of the present study. 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Cite Share Download PDF Status: Published Journal Publication published 20 Dec, 2025 Read the published version in BioNanoScience → Version 1 posted Editorial decision: Revision requested 02 Sep, 2025 Reviews received at journal 01 Sep, 2025 Reviews received at journal 26 Aug, 2025 Reviews received at journal 23 Aug, 2025 Reviewers agreed at journal 22 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers invited by journal 19 Aug, 2025 Editor assigned by journal 19 Aug, 2025 Submission checks completed at journal 17 Aug, 2025 First submitted to journal 28 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7233568","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":505774327,"identity":"2bf08a56-952a-4156-86da-3bfc82b5eb24","order_by":0,"name":"Babay A. Suliasih","email":"data:image/png;base64,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","orcid":"","institution":"Universiti Kebangsaan Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Babay","middleName":"A.","lastName":"Suliasih","suffix":""},{"id":505774328,"identity":"14b6fa3a-22de-47bf-977e-1414468cfa1e","order_by":1,"name":"Anis Sakinah","email":"","orcid":"","institution":"Universitas Negeri Jakarta","correspondingAuthor":false,"prefix":"","firstName":"Anis","middleName":"","lastName":"Sakinah","suffix":""},{"id":505774329,"identity":"e117a3d3-2caf-45f0-9c6d-f35aed043f64","order_by":2,"name":"Marissa Angelina","email":"","orcid":"","institution":"National Research and Innovation Agency (BRIN)","correspondingAuthor":false,"prefix":"","firstName":"Marissa","middleName":"","lastName":"Angelina","suffix":""},{"id":505774330,"identity":"cacc8583-8288-4806-99a9-d36240117d95","order_by":3,"name":"Haliza Katas","email":"","orcid":"","institution":"Universiti Kebangsaan Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Haliza","middleName":"","lastName":"Katas","suffix":""},{"id":505774331,"identity":"cd441353-9bc3-42c5-b3f9-c5bbe7d4247d","order_by":4,"name":"Setia Budi","email":"","orcid":"","institution":"Universitas Negeri Jakarta","correspondingAuthor":false,"prefix":"","firstName":"Setia","middleName":"","lastName":"Budi","suffix":""}],"badges":[],"createdAt":"2025-07-28 11:53:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7233568/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7233568/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12668-025-02273-y","type":"published","date":"2025-12-20T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89988628,"identity":"089091a3-7c85-4c81-995f-5614222aa29c","added_by":"auto","created_at":"2025-08-27 07:04:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":349730,"visible":true,"origin":"","legend":"\u003cp\u003eXRD diffraction patterns of AuNPs electrodeposited with different scan rates\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7233568/v1/999c8e553e461c1301d8ce28.png"},{"id":89988604,"identity":"d5c8c1d8-56cb-45ae-b3f1-fa1cbc42548a","added_by":"auto","created_at":"2025-08-27 07:04:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":165624,"visible":true,"origin":"","legend":"\u003cp\u003eEDX spectrum of AuNPs\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7233568/v1/a98cce04d3777f93c04d99b7.png"},{"id":89988605,"identity":"89013e8e-99fe-4163-849b-f8a96519feed","added_by":"auto","created_at":"2025-08-27 07:04:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":910398,"visible":true,"origin":"","legend":"\u003cp\u003eSEM analysis of AuNPs synthesized at various scan rates of (A) 80 mv/s, (B) 125 mV/s, (C) 250 mV/s, (D) 300 mV/s, and 375 mV/s\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7233568/v1/a459c970642fb009bddef184.png"},{"id":89990433,"identity":"803c6f7c-2d13-49e2-b55e-e1d0bf172f94","added_by":"auto","created_at":"2025-08-27 07:12:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":472532,"visible":true,"origin":"","legend":"\u003cp\u003eSpectrum UV–vis spectra of control and AuNPs samples synthesized at different scan rates with lmax at 514 nm of (A) 375 mV/s, (B) 300 mV/s, (C) 250 mV/s, (D) 125 mV/s. and (E) 80 mV/s\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7233568/v1/4d5924c7b422227c85b4d8ba.png"},{"id":89988614,"identity":"167d6287-6fc4-4684-ab3a-09d7ab231ae6","added_by":"auto","created_at":"2025-08-27 07:04:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29347,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between the scan rate and the percent inhibition in the DPPH method\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7233568/v1/32478b8e07ab6ca657c328cb.png"},{"id":89988617,"identity":"b0ae01fa-e264-446e-bb50-70b193e4e5a3","added_by":"auto","created_at":"2025-08-27 07:04:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":187590,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of DPPH free radicals reduction by AuNPs\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7233568/v1/68fe7b9658a74352107f25d5.png"},{"id":89990439,"identity":"44ec784d-d815-4eef-9996-d7e13d6c0d31","added_by":"auto","created_at":"2025-08-27 07:12:42","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":11860,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between scan rate and percent reduction in the FRAP test and percent inhibition in the DPPH test\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7233568/v1/1ba6caedf2b0d24621b10341.jpg"},{"id":98813821,"identity":"21c8f632-0a4b-42f1-b077-6b07a1137689","added_by":"auto","created_at":"2025-12-22 16:00:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2883520,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7233568/v1/111bec98-4529-4d26-9d1f-3bbd8a59d592.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Size-Dependent Antioxidant Effects of Gold Nanocrystalline Films","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFree radicals are molecular species that possess at least one unpaired electron in their outer shell. These species can arise as a result of the oxidative phosphorylation respiratory cycle, which can interact with biological macromolecules including cellular DNA, leading to single-strand and double-strand breaks that ultimately can initiate cellular aging, mutagenic alterations, cardiovascular disorders, and the development of tumors or cancer (Jomova \u003cem\u003eet al\u003c/em\u003e. 2023). Oxidative stress refers to the physiological state that occurs when an organism\u0026rsquo;s internal defence mechanisms, encompassing enzymatic, non-enzymatic, and dietary components, are inadequate to counteract an excessive buildup of free radical species.\u003c/p\u003e\u003cp\u003eThis condition leads to the oxidative damage to both extracellular and cellular macromolecules, such as lipids, proteins, and nucleic acids, ultimately resulting in tissue injury (Chen \u003cem\u003eet al\u003c/em\u003e. 2024). However, in general, these free radical reactions can be inhibited or mitigated by certain antioxidant compounds obtained from natural or synthetic sources [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAntioxidants are defined as compounds that possess the ability to delay, regulate, or inhibit the process of autoxidation (Chukiatsiri \u003cem\u003eet al\u003c/em\u003e. 2024). They play a crucial role in human health by impeding or delaying undesired oxidation reactions, thereby preventing oxidative stress associated with diseases such as hypertension, neurodegenerative disorders, or cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Both natural and synthetic antioxidants have limited effectiveness due to poor absorption, difficulty in entering cells, and breakdown during delivery. However, the current progress in nanotechnology has facilitated the development of a variety of inorganic nanoparticles that exhibit enhanced antioxidant properties, thus offering promising potential for various applications (Khalil \u003cem\u003eet al\u003c/em\u003e. 2020, [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eOne of the nanomaterials that can function as an antioxidant is gold nanoparticles (AuNPs). AuNPs have been extensively employed in the recent research due to their unique chemical and physical properties (Dong \u003cem\u003eet al\u003c/em\u003e. 2024). Studies show that AuNPs act as antioxidants, demonstrating strong kinetic effects in scavenging of reactive oxygen species (ROS) within living cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The kinetic effect is greatly affected by the size of AuNPs. Smaller particles have been shown to enhance catalytic activity due to their ability to generate a larger surface area, facilitating a greater number of active sites available for interaction with free radicals (A. Bano \u003cem\u003eet al\u003c/em\u003e. 2023; Suliasih \u003cem\u003eet al\u003c/em\u003e., 2024). Moreover, numerous studies have shown that AuNPs possess a notable antioxidant effect, highlighting their potential in medical applications including wound healing, anti-asthma, antipyretic, anti-cancer and antidiabetic agents. Additionally, the relatively low toxicity of AuNPs in human subjects increases their desirability for use in therapies [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn antioxidant research, AuNPs are primarily synthesized through biosynthesis methods [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This approach mainly utilizes bioactive phytochemical compounds with antioxidant properties to reduce gold ions and form AuNPs. Since the antioxidant activity exhibited by this AuNPs do not originate solely from the AuNPs themselves, this study aimed to determine the antioxidant activity response of AuNPs themselves [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For this purpose, AuNPs were produced as a solid phase or thin film without employing any antioxidant compounds in their synthesis using electrodeposition method. Electrodeposition is known as a facile and low-cost method to directly grow metallic deposit in a form of uniform films [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This allows the observation of the mechanism of single AuNPs as antioxidants. This study is aiming to determine the effects of gold particle size on their antioxidant activities by varying the scanning rate of the electrodeposition process.\u003c/p\u003e"},{"header":"Materials and Method","content":"\u003cp\u003eThe materials used in this study were gold(III) chloride trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO), sulphuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), 2,2-diphenyl-1-picrylhydrazyl (DPPH) ethanol, sodium acetate trihydrate, distilled water, TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), hydrochloric acid, ferric chloride, trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), Fluorine doped Tin Oxide (FTO) glass as the substrate, and 24-well microplates as the sample containers for testing.\u003c/p\u003e\n\u003cp\u003eSynthesis of AuNPs\u003c/p\u003e\n\u003cp\u003eThe synthesis of AuNPs was carried out in the electrolyte solution containing 0.5 mM gold solution (HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO) dissolved in 0.5 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. By employing the cyclic voltammetry method, AuNPs were grown with a starting voltage of -1.0 V vs Ag/AgCl and a final voltage of 1.5 V vs Ag/AgCl. The scan rate variations employed in the study were 375, 300, 250, 125, and 80 mV/s. This electrochemical deposition was performed in a three-electrode cell with Ag/AgCl acted as the reference electrode, FTO served as the working electrode, and a platinum plate was functioned as the counter electrode. The experimental procedures were conducted at 25\u0026deg;C. Once the synthesis was finished, the deposited AuNPs on the substrate were subjected to rinsing by distilled water, and subsequently dried.\u003c/p\u003e\n\u003cp\u003eCharacterization\u003c/p\u003e\n\u003cp\u003eThe structural analysis of the electrodeposited AuNPs was conducted using a SMARTLAB Rigaku X-ray diffractometer (XRD) with Cu-K-Alpha1 radiation (\u0026lambda;\u0026thinsp;=\u0026thinsp;1.540598 \u0026Aring;). A Scanning Electron Microscope (Thermo Scientific: Quanta 650) and an Energy Dispersive X-Ray Spectrometer (Qxford Instrument: Xplore 15) were employed to investigate the morphological and elemental analysis, respectively. The particle size distribution of the deposited AuNPs was determined from the obtained SEM micrographs using ImageJ software.\u003c/p\u003e\n\u003cp\u003eAntioxidant determination\u003c/p\u003e\n\u003cp\u003eThe antioxidant activity response of AuNPs was evaluated using in vitro test methods specifically the DPPH and Ferric Reducing Antioxidant Power (FRAP).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eDPPH Assay\u003c/h2\u003e\n \u003cp\u003eThe absorbance of samples was measured by a Spectrophotometer (Microplate Reader Thermo Scientific: Elisa Reader). Deposited AuNPs (dimension of 5 mm x 15 mm) were distributed into a 24-well microplate and subsequently submerged in 1.3 ml of 100 \u0026micro;M DPPH-ethanol solution. Afterward, the samples were subjected to agitation through shaking and the absorbance was measured using a spectrophotometer at 300\u0026ndash;750 nm at the pre-determined time intervals: 15, 30, 45, 60, 75, 90, 105, 120, 135, and 150 minutes. The percentage inhibition of DPPH was determined using Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\:\\%Inhibition=\\frac{Control\\:Absorbance-Sample\\:Absorbance\\:}{Control\\:Absorbance\\:}\\:x\\:100\\%$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eThe control absorbance is the peak absorbance exhibited by the DPPH solution, and the sample absorbance is the peak absorbance seen in the DPPH solution after adding AuNPs samples at a wavelength of 518 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eFRAP\u003c/h3\u003e\n\u003cp\u003eThe sample was prepared similarly to the DPPH test. The FRAP solution consisted of sodium acetate trihydrate (0.3 M; pH 3.6)\u0026thinsp;+\u0026thinsp;TPTZ (2,4,6-tris(2-pyridyl)-s-triazine (10 mM/L)) in 40 mM HCl\u0026thinsp;+\u0026thinsp;ferric chloride (20 mM) with a ratio of 10:1:1, and then its absorbance was measured at a wavelength of 596 nm. To measure the antioxidant activity of AuNPs in reducing Fe3+, the Eq.\u0026nbsp;(2) was used.\u003c/p\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:\\%Reduction=\\frac{Sample\\:Absorbance-Control\\:Absorbance\\:}{Sample\\:Absorbance\\:}x\\:100\\%$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eThe sample absorbance refers to the absorbance value of the FRAP solution after the addition of AuNPs samples, while the control absorbance represents the maximum absorbance value of the FRAP solution.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eCharacterization\u003c/p\u003e\n\u003ch3\u003eXRD\u003c/h3\u003e\n\u003cp\u003eThe XRD results of the AuNPs electrodeposited using cyclic voltammetry technique with different scan rate are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea. The peaks of Au crystal appeared at 2\u0026theta; 38.32\u0026deg;, 44.45\u0026deg;, 64.68\u0026deg;, and 77.72\u0026deg; that could be associated to the reflection from (111), (020), (022) and (131) planes, respectively. This pattern is agreed with the crystal open database (COD) No. 96-901-1614 for metallic Au phase. Though, the peaks at 2\u0026theta; 38.32\u0026deg; was observed to be overlap with the peak of fluor tin oxide substrate at 2\u0026theta; 37.87\u0026deg;. However, the magnified patterns, which are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb, indicate the peaks broadening and shifting to higher 2\u0026theta; clearly confirm that the reflection from (111) plane of Au was observed. In this case, the high peaks of the substrate were due to the very thin Au films that allow high intensity reflection from the substrate. The crystallite size of Au calculated by following Scherrer Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) indicates that the fine crystallites ranging from 19 to 31 nm were successfully grown by this approach.\u003c/p\u003e\n\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e$$\\:D=\\frac{K{\\lambda\\:}}{\\beta\\:\\text{cos}\\theta\\:}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eWhere D is crystallite size, K is shape factor, \u0026lambda; is the X-ray radiation wavelength, \u0026beta; is peak broadening, and \u0026theta; is the Bragg angle.\u003c/p\u003e\n\u003ch3\u003eEDX \u0026amp; SEM\u003c/h3\u003e\n\u003cp\u003eThe characterization results using EDX confirm the existence of Au element on the layer of the substrate. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e presents the spectrum signals of AuNPs ranging from 2.1 to 12.7 keV. In addition to the presence of Au, spectra corresponding to other elements from the FTO substrate are also observed, namely Sn and Si.\u003c/p\u003e\n\u003cp\u003eThe results of SEM for AuNPs synthesized with varying scan rates are depicted in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The resulting particle morphology ranges from spherical to irregular, with some particles overlapping and merging to form large nanoclusters. As the scan rate was gradually increased, the particle size appeared to be decreased while the number of particles appeared to be increased. Subsequently, the number of nanoclusters decreased, resulted in well dispersed nanoparticles.\u003c/p\u003e\n\u003cp\u003eThe SEM images were then processed to ImageJ software for determining its particle size as depicted in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The particle size results indicate that the faster scan rates produced finer particles compared to that of slower scan rates. This phenomenon arises because increasing the scan rate shortens the particle growth phase, leading to the formation of smaller nanoparticles [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Conversely, slower scan rates yield larger AuNPs due to the prolonged particle growth, which increases the likelihood of particle overlap, resulting in agglomeration and subsequent enlargement of particles.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eParticle size resulting from variations in scan rate\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScan Rate (mV/s)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParticle Size (nm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e78.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e66.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eAntioxidant activity test\u003c/p\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eDPPH\u003c/h2\u003e\n \u003cp\u003eThis DPPH assay is the simplest and most widely used method for determining antioxidant activity. It involves the conversion of free radical into non-radical species through the presence of antioxidant compounds [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates a decrease in DPPH absorbance values across all AuNPs samples. The data clearly show a consistent decline in absorbance over the incubation period for each sample. This trend signifies the ability of AuNPs to inhibit DPPH. The level of DPPH inhibition was quantified as a percentage, providing a metric for evaluating the antioxidant efficacy of AuNPs.\u003c/p\u003e\n \u003cp\u003eThe data in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e indicates that the sample prepared at the scan rate of 300 mV/s exhibits the highest inhibition activity of 43.91%. This result correlates with the particle size, as antioxidant activity increases with higher scan rates. As previously mentioned, the particle size obtained at this scan rate was 66.8 nm, which is lower than that achieved at lower scan rates (80 mV/s to 250 mV/s). These smaller particles result in a large of surface area, contributing to increased antioxidant activity. However, an anomaly is observed for the sample produced at 375 mV/s, where the inhibition is lower than that of the 300 mV/s sample but still higher than the samples produced at 250 mV/s, 125 mV/s and 80 mV/s. This phenomenon may be attributed to the reduced number of particles formed on the substrate, as illustrated by the SEM micrograph (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee). These findings indicate that the optimal active sites for reaction with DPPH were achieved at a scan rate of 300 mV/s.\u003c/p\u003e\n \u003cp\u003eAuNPs can donate electrons to unpaired DPPH radicals, reducing DPPH to form stable non-radical compounds [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. The proposed quenching mechanism involves electron transfer from AuNPs to stabilize the nitrogen atoms in DPPH by forming coordinated covalent bonds. This observation leads to the formation of a coordination covalent bond between gold and nitrogen (Au-N), which effectively quenches the initially radical DPPH compound. The stabilization of the nitrogen atoms by the gold atoms is a significant factor in this process [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. The quenching process of free DPPH radicals is characterized by a colorimetric shift from purple to yellow, which signifies the conversion of free DPPH radicals into stable compounds. Figure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e shows an illustration of the assumed mechanism of quenching free DPPH radicals by AuNPs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eFRAP\u003c/h2\u003e\n \u003cp\u003eThe FRAP method is used to validate the findings of AuNPs samples identified by the DPPH method. Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the sample prepared at a scan rate of 300 mV/s exhibited the highest reduction percentage, reaching 38.22%. The findings indicate a notable rise in absorbance in the FRAP samples that immersed with AuNPs/FTO.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe correlation between the scan rate and the percentage of inhibition in the FRAP test\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReduction (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e80 mV/s\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16,10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e125 mV/s\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16,14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e250 mV/s\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17,88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e300 mV/s\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38,22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e375 mV/s\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35,00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThis phenomenon may demonstrate a reduction reaction of Fe\u003csup\u003e3+\u003c/sup\u003e complex compounds to Fe\u003csup\u003e2+\u003c/sup\u003e complex compounds as a result of electron donation from AuNPs [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. This observation provides evidence for the presence of antioxidant activity. The findings are consistent with the analysis carried out utilizing the DPPH method. When correlated with SEM data, it is suggested that the sample scanned at a rate of 300 mV/s showed the highest levels of inhibition and reduction, due to its abundance of spherical to irregular-shaped particles. It is hypothesized that the irregular-shaped particles contain many active sites, edges, or corners, leading to increased conductivity [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] and displaying greater surface reactivity [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], ultimately enhancing their effectiveness as antioxidants.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e depicts the correlation between scan rate and reduction percentage in the FRAP test, as well as inhibition percentage in the DPPH test. The findings in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e indicate that the reduction percentage obtained via the FRAP method is comparatively lower than that observed through the DPPH method. The suggests that the AuNPs synthesized using the FRAP method may have suboptimal properties for reducing FeIII-TPTZ complexes under thermodynamic conditions, resulting in slower reaction rates [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, these AuNPs are highly effective at quenching DPPH radicals, leading to the highest inhibition values observed with the DPPH method.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAuNPs synthesized through the electrodeposition method at different scan rates, demonstrate considerable antioxidant potential. This is evidenced by their capacity to interact with DPPH radicals and to reduce Fe\u003csup\u003e3+\u003c/sup\u003e ions effectively. Specifically, AuNPs synthesized at a scan rate of 300 mV/s were shown to inhibit DPPH free radicals by 43.91% and reduce Fe\u003csup\u003e3+\u003c/sup\u003e ions by 38.22%. The observed increase in antioxidant activity is likely attributable to the reduced particle size of the AuNPs. This reduction in size increases the available surface area and the density of the active sites, thereby promoting more efficient radical scavenging and ion reduction processes. These findings underscore the remarkable antioxidative properties of AuNPs, particularly those synthesized under specific conditions, highlighting their promising role in combating oxidative stress-related disorders. The irregular morphology of these AuNPs enhances their surface area, facilitating greater interaction with reactive species and enhancing their antioxidant efficacy. Furthermore, the optimal synthesis conditions, as demonstrated by the selected scan rate, highlight the critical significance of meticulous control over experimental parameters in the customization of the properties of AuNPs for targeted applications. These results pave the way for further exploration and utilization of AuNPs as potent antioxidants in various biomedical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests.\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics Declaration.\u003c/h2\u003e\u003cp\u003eNot Applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.A.S.: Conceptualisation, Methodology, Investigation, Data Analysis, Writing_original draft, Writing_review \u0026amp; editing; A.S: Investigation, Data Analysis, Project Administration; M.A.: Resources, Supervision; H.K.: Conceptualisation, Methodology, Resources, Supervision, Writing_Review \u0026amp; Editing, Funding Acquisition; S.B: Conceptualisation, Methodology, Resources, Funding Acquisition, Supervision, Writing_Review \u0026amp; Editing, All authors have read and approved the final manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge the financial support provided by the Lembaga Penelitian dan Pengabdian Masyarakat (LPPM) at Universitas Negeri Jakarta through the International Collaborative Research scheme, under contract number: 19/KI/LPPM/III/2025, and Universiti Kebangsaan Malaysia through a research grant (Geran Universiti Penyelidikan UKM (GUP-2022-004)\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eNo datasets were generated or analysed in the course of the present study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJomova, K., Raptova, R., Alomar, S. 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Proc.\u003c/em\u003e 2370 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/5.0062364\u003c/span\u003e\u003cspan address=\"10.1063/5.0062364\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"gold nanoparticle, antioxidant, electrodeposition, film, particle size","lastPublishedDoi":"10.21203/rs.3.rs-7233568/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7233568/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe exceptional chemical and physical properties of gold nanoparticles (AuNPs) have led to their significant use in both medicinal and cosmetic applications. This study investigated the antioxidant properties of AuNPs. Various sizes of AuNPs were synthesized through the electrodeposition technique, resulting in the formation of deposited films. Structural analysis with an X-ray diffractometer (XRD) revealed that a face-centered cubic structure of metallic gold with fine crystallite size (19 to 31 nm) was successfully formed on the substrate. Morphological studies employing a field emission scanning electron microscope (FESEM) showed that the deposited film consisted of relatively fine particles, providing numerous active sites for the radical scavenging mechanism. Antioxidant assays demonstrated that the deposited film exhibited promising antioxidant activity, particularly with a large number of very fine gold particles. This efficacy was evidenced by their efficacy in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, with a maximum percentage inhibition of 43.91%, and the Ferric Reducing Antioxidant Power (FRAP) assay, with a maximum percentage reduction of 38.22%. These results highlight the notable antioxidative capabilities of AuNPs, especially those produced under specific experimental conditions. This suggests their potential in tackling problems related to oxidative stress.\u003c/p\u003e","manuscriptTitle":"Size-Dependent Antioxidant Effects of Gold Nanocrystalline Films","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 07:04:35","doi":"10.21203/rs.3.rs-7233568/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-02T06:40:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-01T11:34:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-26T10:44:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-23T13:20:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205268177969597515685261500882992767664","date":"2025-08-22T09:15:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"186772113075708363546869472802675982658","date":"2025-08-19T06:29:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61598300695337742305697441512966590292","date":"2025-08-19T05:29:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-19T05:26:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-19T05:18:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-18T01:21:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-07-28T11:41:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e4c213be-9d98-41ca-b7bb-2c54beed330e","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T15:58:59+00:00","versionOfRecord":{"articleIdentity":"rs-7233568","link":"https://doi.org/10.1007/s12668-025-02273-y","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2025-12-20 15:56:57","publishedOnDateReadable":"December 20th, 2025"},"versionCreatedAt":"2025-08-27 07:04:35","video":"","vorDoi":"10.1007/s12668-025-02273-y","vorDoiUrl":"https://doi.org/10.1007/s12668-025-02273-y","workflowStages":[]},"version":"v1","identity":"rs-7233568","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7233568","identity":"rs-7233568","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}