Development of a Silver Screen-Printed Paper Card Modified Gold Nitrogen Doped Graphene Electrode for Voltammetric Determination of Tenofovir

Development of a Silver Screen-Printed Paper Card Modified Gold Nitrogen Doped Graphene Electrode for Voltammetric Determination of Tenofovir | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of a Silver Screen-Printed Paper Card Modified Gold Nitrogen Doped Graphene Electrode for Voltammetric Determination of Tenofovir Magaji Ladan, Salamatu Hayatu, Abdulrahman A Audu, Musa Babashani, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8253957/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract A new method for detecting tenofovir has been developed using a silver screen-printed paper card and voltammetric techniques. Gold nanoparticles (Au) were deposited onto nitrogen-doped graphene (NG) using a simple reduction method, resulting in a gold-decorated nitrogen-doped graphene (Au-NG) composite. The paper card was screen printed with silver ink, after which the composite was used to modify the paper card-based silver electrode and applied in the voltammetric detection of tenofovir. The sensor's characteristics were analysed using transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX). Electrochemical studies were performed using cyclic voltammetry (CV) and Differential pulse voltammetry (DPV). DPV with the modified electrode demonstrated a sensitivity 1.6 times higher than that of the unmodified electrode, achieving a linear calibration at a scan rate of 100 mV/s and pH 8 in acetate buffer. The sensor displayed strong resistance to interference from other ions and was successfully tested on real samples. gold nitrogen-doped graphene tenofovir paper card screen-printed silver electrode voltammetry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Tenofovir is a widely utilized antiretroviral medication classified as a nucleotide reverse transcriptase inhibitor (NRTI), commonly prescribed for the treatment of HIV [ 1 , 2 ] and chronic hepatitis B infections [ 3 , 4 ]. However, it is crucial to monitor its levels in the human body and the environment due to the potential risk of kidney and liver complications [ 2 – 4 ]. Consequently, developing advanced, cost-effective, and precise sensing systems for determining tenofovir concentrations in real samples is essential [ 5 ]. Given its extensive clinical application and the risk of environmental contamination from pharmaceutical waste, accurate and sensitive methods for detecting and quantifying tenofovir in both pharmaceutical and ecological samples are vital [ 6 – 8 ]. The effectiveness of tenofovir disoproxil fumarate in managing HIV infections underscores the importance of quality control monitoring for this drug in its formulation and dissolution. Recently, tenofovir has been detected in bulk samples, pharmaceutical preparations, and tablet dosage forms utilizing a screen-printed carbon electrode. which serves as the first solid contact polymeric potentiometric sensor embedded with calix[ 6 ]arene as a supramolecular ionophore. The incorporation of a graphene nanocomposite layer significantly enhanced the sensor's performance and stabilized the potential signal compared to a control sensor, achieving a limit of detection (LOD) of 7.3 µM [ 7 ]. Additionally, a low-cost point-of-care assay was developed to assess tenofovir levels in urine accurately. Designed to support PrEP (pre-exposure prophylaxis) counseling, this urine tenofovir assay has shown improvements in both short-term and long-term adherence metrics, thus holding promise for enhancing long-term PrEP adherence [ 8 ]. Graphene, a remarkable two-dimensional nanocarbon material, inspires global interest for its potential to transform applications such as solar cells, fuel cells, supercapacitors, and sensors [ 9 ]. Graphene possesses several unique qualities, making it the best substrate for dispersing noble metal nanoparticles (NPs). These remarkable qualities, superb chemical and thermal stability, a vast surface area, and exceptional electrical conductivity, empower us to achieve extraordinary things [ 10 ]. Doping graphene with foreign atoms, like nitrogen, could improve its electrochemical performance and reactivity. Furthermore, compared to undoped graphene, incorporating nitrogen atoms into the lattice increases the active sites, creating a harmonious environment for a more even distribution of smaller metal nanoparticles [ 11 ]. Noble metal nanoparticles such as Au, Ag, and Pt were incorporated onto NG to develop new nanocomposites with enhanced electrochemical, electrocatalytic, and stability properties. There is a strong binding force between AuNPs and NG because of their high active surface area and superior catalytic capabilities [ 10 – 13 ]. Voltammetric techniques, particularly electroanalytical methods such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV), have gained attention as powerful tools for drug analysis [ 14 – 16 ]. The voltammetric technique enables the qualitative and quantitative determination of analytes at low concentrations with high specificity. These characteristics make the voltammetric technique suitable for drug confirmation [ 15 ]. Electrochemical methods are instrumental in taking simultaneous measurements of multiple substances at once, and voltammograms can be displayed for different substances simultaneously [ 17 ]. These techniques also offer high sensitivity, selectivity, low detection limits, and cost-effectiveness, making them suitable for determining tenofovir in complex matrices [ 18 – 23 ]. The ability of voltammetry to detect electroactive species based on their redox behavior allows for the precise analysis of tenofovir without the need for extensive sample preparation [ 18 – 24 ]. Numerous studies have demonstrated the ability to detect tenofovir through electrochemical methods [ 5 , 7 , 18 – 24 ]. Some electrochemical sensors, developed with gold or carbon-based nanomaterials [ 5 , 20 ], exhibit extremely high sensitivity, swift detection, and can be employed as field-deployable point-of-care (POC) devices [ 25 ]. Among the different fabrication techniques used for drug detection, certain methods necessitate the incorporation of valves into rigid substrates such as glass and silicon, which involves many steps in both production and operation. This challenge can be addressed by using a paper substrate, which has proven to be an effective material in voltammetry, as it is currently the most cost-effective option and requires fewer steps in fabrication and operation [ 16 ]. This article investigates the application of a paper card substrate in creating a screen-printed electrode for the differential pulse voltammetric detection of tenofovir, examining the electrochemical behavior toward the drug and various experimental parameters that affect its detection. Experimental Materials Chemicals All chemicals, materials, and reagents used in this study were of analytical grade. Nitrogen-doped graphene, chloroauric acid (HAuCl4), and silver conductive ink were sourced from Sigma Aldrich (USA). Sodium acetate and acetic acid were obtained from Kermel Chemicals (China). Potassium ferricyanide and potassium chloride were purchased from Harris Reagent (England), and KCl.Ag was acquired from Radiometer Analytical (France). HmbG Chemicals supplied Lead (II) chloride, L (+)-ascorbic acid, and urea. Uric acid, hydrochloric acid (HCl), and hydrazine hydrate were obtained from Sigma Aldrich (Germany). Sodium chloride, glucose, and citric acid were sourced from R&M Chemicals (UK), and phosphate buffer was obtained from Bendosen Laboratory Chemicals (Malaysia). Pure tenofovir powder was provided by Aminu Kano Teaching Hospital (AKTH), and the metallic gold card used in this research was also of analytical grade. Experimental Measurement The morphology and dimensions of NG and Au-NG were characterized through Transmission Electron Microscopy (TEM) employing a JEOL JEM-2100F instrument. To evaluate the elemental composition and spectral characteristics of NG and Au-NG, Energy Dispersive X-ray Spectroscopy (EDX) was performed using a JEOL JSM 7600F Field Emission Scanning Electron Microscopy (FESEM) equipped with EDX capabilities. Additionally, electrochemical measurements were executed with an Autolab potentiostat operated via Nova software version 2.1.4. The potentiostat was coupled to a computer and the gold nitrogen-doped graphene screen-printed paper electrode (Au-NGSPE). CV of potassium ferricyanide was obtained by scanning potential from − 0.2 to 0.9 V at 50 mVs -1 , while DPV for the study of tenofovir was scanned from 0.0 to 0.5 V at a scan rate of 100 mVs -1 . Synthesis of Gold Nitrogen Doped Graphene (Au-NG) A 2 mL suspension of NG (concentration: 10 mg/mL) was mixed with 20 mL of a 0.05 M aqueous solution of HAuCl 4 . Sodium hydroxide (0.1 mol/L) was then added to adjust the pH to 8. After heating the mixture to 80°C for 35 minutes under constant stirring, 0.01 mL of hydrazine monohydrate (NH 2 NH 2 .H 2 O) was introduced. To facilitate the decoration of the NG nano sheets with gold, the mixture was allowed to react for an additional 30 minutes [ 10 ]. Device Design A three-electrode layer was screen printed with silver ink onto a paper card, then cured for 30 minutes at 120°C [ 26 ]. Following this, individual electrodes were cut out as shown in Fig. 1 and stored in a sealed bag. The Au-NGSPE was assembled by drop-coating the surface of the working electrode with 20 µL of Au-NG, and allowing the solution to dry at room temperature before use [ 27 ]. Results Transmission Electron Microscopy Figure 2 A shows the image of NG displaying the sheet like structure of graphene, while Fig. 2 B illustrates the sheet like structure of the NG with Au nanoparticles in the size range of 70 nm to 80 nm decorated on it. The number of Au nanoparticles appears limited, likely due to the synthesis method and the ratio of NG to Au used in the process. Energy Dispersive X-Ray (EDX) Spectroscopy Figure 3 A presents the spectral image of NG, where the peaks of carbon, nitrogen, and oxygen are clearly visible, accompanied by their percentage weights. The sharp peak indicates that carbon constitutes the highest amount at 82.59% in NG. Figure 3 B shows the spectral image of Au-NG, revealing peaks for carbon, oxygen, gold, and silver, along with their percentage weights. In this case, carbon still dominates at 68.8%, followed by gold at 20.05%. The absence of a nitrogen peak suggests a reaction between the nitrogen component and the gold. Figure 3 C displays the spectral image of Au-NG in combination with tenofovir, where peaks for carbon, oxygen, gold, and silver are again observed, along with their percentage weights. Figure 3 B and 3 C exhibit notable differences in their elemental composition. In Fig. 3 B, the concentration of carbon is measured at 68.8%, alongside Au content of 20.05%. In contrast, Fig. 3 C shows a significant reduction in both carbon and gold percentages. Specifically, the amount of carbon is lower. However, the most striking change is observed in the oxygen content, which has dramatically increased from 8.47% in Fig. 3 B to an impressive 38.53% in Fig. 3 C. This increase in oxygen suggests a substantial alteration in the chemical makeup or the environmental conditions impacting the samples represented by these Figures. Electrochemical Response As shown in Fig. 4 , CV in 2 mM ferricyanide and 0.1 M KCl gave an anodic and cathodic peaks for the reduction and oxidation of ferricyanide [Fe(CN) 6 ] 3− to ferrocyanide [Fe(CN) 6 ] 4− . The unmodified SPE shows no definite reduction and oxidation peaks, while the Au-NGSPE showed a reduction peak around 0.4 V and an oxidation peak around 0.2 to 0.3 V, which agrees with the literature [ 28 – 29 ]. Effect of pH The pH gives information about the redox behavior of the detection such as, sensitivity. When increase in pH is proportional to increase in current, it means the component is better oxidized or reduced at basic pH. From the pH study in Fig. 5 , tenofovir is better oxidized at basic pH than acidic pH with Au-NGSPE. The increase in peak current is due to deprotonation [ 30 ]. The pH rises from pH 4 to 6, with a sharp decrease at pH 7 but gradually increases up to pH 10. The electro-oxidation of tenofovir is high at a basic pH. Therefore, pH 8 was chosen as the optimum pH for the detection of both tenofovir as the real sample analysis will be tested in urine sample, and both body pH and urine pH fall between the range of 7 to 8. Differential Pulse Voltammetry The detection sensitivity was studied under DPV scan for both the unmodified electrode (UMSPE) and the Au-NGSPE in acetate buffer containing tenofovir. The Au-NGSPE exhibited a significant enhancement in the current measurements, as shown in Fig. 6 . Therefore, different concentrations of tenofovir ranging from 20 µM to 100 µM were investigated using DPV in 0.1 M acetate buffer at pH 8 and a scan rate of 100 mV/s. The results produced a calibration plot with an excellent linear regression value (R² = 0.9909) for the Au-NGSPE as displaced in Fig. 7 . The detection of tenofovir using Au-NGSPE achieved a limit of detection (LOD) of 16.99 µM. Validation and Real (Urine) Sample Application The proposed method’s applicability was evaluated through the analysis of tenofovir in urine samples. The results for both the original samples and those spiked with tenofovir were validated using gas chromatography-mass spectrometry (GC-MS) analysis. Table 1 demonstrates a strong correlation between the newly developed analytical method and the established standard method, indicating that the proposed approach is suitable for tenofovir determination. Table 1 Determination of Tenofovir in urine sample Sensor Tenofovir Added (µM) Tenofovir Found (µM) % Recovery Au-NG/SPE Tenofovir 16 (LOD) 30 33 110 200 160 80 GC-MS 30 25 83.3 200 170 85 Discussion The results indicated that the application of Au-NG significantly enhanced the sensitivity of the screen-printed electrode (SPE). This improvement is attributed to the synergistic effect between gold nanoparticles (AuNPs) and reduced graphene oxide (NG) [ 11 ]. Additionally, the deposition of gold on the surface of NG increases charge transfer, leading to greater electrocatalytic activity and facilitating the electrochemical oxidation of tenofovir on the Au-NG/SPE surface. [ 10 , 31 ]. Tenofovir can interact with gold nanoparticles on the surface of nitrogen-doped graphene through coordination bonding. The nitrogen-doped graphene can serve as a catalyst for the oxidation of tenofovir, leading to the formation of reactive oxygen species. Tenofovir can attach onto the surface of AuNPs through the amino group by electrostatic interaction and through the reactive oxygen species to give the oxidized tenofovir. As such, the more stable amine-reactive intermediates group of tenofovir can link with gold nanoparticles through the oxygen hybrid ring and hydroxyl group [ 31 ] as illustrated in Scheme 1 . Both EDX and TEM analyses confirm the attachment of gold to the surface of NG; however, the amount of gold present is low compared to NG. A study investigating the variation of NG and Au was not conducted. There is potential to achieve significantly greater sensitivity by performing this study and by modifying the synthesis method, such as by introducing a different reducing agent. The calibration produced a strong linear plot. However, significant potential variations were observed when the linear range was expanded beyond the current limits. This resulted in high absorption on the electrode surface, which led to electrode damage and sample contamination. Foreign ions were selected based on those typically found in urine. The study found that these foreign ions did not significantly impact the detection of tenofovir in Table 2 . The highest levels of interference were observed with glucose and urea. Ascorbic acid showed slight interference, while Na+, and K + had negligible effects, and uric acid demonstrated no interference at all. This suggests that all the ions have a broader potential range in conjunction with tenofovir, but the interference potential of glucose and urea may be more similar to that of tenofovir. Table 2 Interference Study for 100µm Tenofovir in the Presence of Foreign Ions Foreign ions Conc(µM) Percentage of Interference (%) Ascorbic Acid 1000 5 Urea 1000 15 Na+ 1000 2.4 K+ 1000 2.4 Glucose 1000 28 Uric Acid 1000 0 Conclusions The Au-NG/SPE was fabricated and applied for voltammetric detection of tenofovir. To the best of our knowledge, this is the first reported use of this approach for tenofovir detection. The results demonstrate that the synthesis method effectively allows for the attachment of gold (Au) onto the surface of the nanographene (NG). Additionally, modifying the screen-printed electrode (SPE) with Au-NG increased the sensitivity of the detection system. Calibration studies conducted for tenofovir demonstrated a robust linear response. The limit of detection was determined to be approximately 16 µM. Overall, the sensor shows promising applicability in the field. Declarations Data Availability Statement : The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Declaration of Competing Interest: The authors declare that they have no competing interests. Consent to Participate declaration : not applicable Ethics declaration : not applicable. Clinical trial number : not applicable Consent to Publish : Not Applicable' as no human study is involved Author Contribution declaration : M.L. and S.H. conceived and designed the study. M.L., A.A.A. and I.T. carried out the experimental work and optimization of the modified gold nitrogen-doped graphene electrode. S.H. performed the electrochemical measurements and analytical validation. M.L., A.B.S and U.S.A. analyzed the data. M.L., S.H. and A.B.S prepared Figures 1–3. 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Schematic illustration of the possible reaction mechanism of citrate capped AuNP and Tenofovir Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 02 Feb, 2026 Reviewers agreed at journal 01 Feb, 2026 Reviews received at journal 30 Jan, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviews received at journal 29 Dec, 2025 Reviewers agreed at journal 21 Dec, 2025 Reviews received at journal 19 Dec, 2025 Reviewers agreed at journal 16 Dec, 2025 Reviewers invited by journal 16 Dec, 2025 Editor assigned by journal 11 Dec, 2025 Submission checks completed at journal 10 Dec, 2025 First submitted to journal 10 Dec, 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. 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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-8253957","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":561362774,"identity":"f94a6d5e-1778-494b-8003-a60a05984f20","order_by":0,"name":"Magaji 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21:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8253957/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8253957/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98758584,"identity":"ee040455-07ad-4981-a52a-7915e38d8cd7","added_by":"auto","created_at":"2025-12-22 09:40:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":467095,"visible":true,"origin":"","legend":"","description":"","filename":"UpdatedManuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/8dcd630445091b21a7904659.docx"},{"id":98758564,"identity":"f5a90378-b84f-497f-afa7-239af470dd23","added_by":"auto","created_at":"2025-12-22 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09:40:25","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89264,"visible":true,"origin":"","legend":"","description":"","filename":"f94f1be427254ad29291e08c00b6ef781structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/415e77e41caf00ac3984a2f1.xml"},{"id":98758615,"identity":"778b0b60-e6fb-402d-a6db-cf51cfab140a","added_by":"auto","created_at":"2025-12-22 09:40:30","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100028,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/105af13d42f6a3b4c1e4e216.html"},{"id":98758627,"identity":"135950a0-f303-4ea6-8f32-9017c79f31e3","added_by":"auto","created_at":"2025-12-22 09:40:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53018,"visible":true,"origin":"","legend":"\u003cp\u003eScreen-printed electrode design\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/9957c9b194fb2fd43cbfbfd0.png"},{"id":98758551,"identity":"75697390-b45d-4948-9a79-ce3c12210ae4","added_by":"auto","created_at":"2025-12-22 09:40:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":175134,"visible":true,"origin":"","legend":"\u003cp\u003eTEM image of (A) NG (B) Au-NG\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/56e95db6376932944935c490.png"},{"id":98758607,"identity":"01076ef8-c9c5-41d1-946d-5e8697aa7888","added_by":"auto","created_at":"2025-12-22 09:40:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47209,"visible":true,"origin":"","legend":"\u003cp\u003eEDX spectra and elemental content of (A) NG (B) Au-NG (C) Au-NG with tenofovir\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/d297013ebeafea19dca556fc.png"},{"id":98758583,"identity":"88ccdbb0-3a5e-4f4d-909f-c4a279b079da","added_by":"auto","created_at":"2025-12-22 09:40:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29435,"visible":true,"origin":"","legend":"\u003cp\u003eCV of the UMSPE and Au-NGSPE in 2 mM Ferricyanide and 0.1 M KCl\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/2255c582dfa8b87a93de7028.png"},{"id":98758590,"identity":"6b4abd96-c3a6-4b84-a240-9d2428eee301","added_by":"auto","created_at":"2025-12-22 09:40:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7226,"visible":true,"origin":"","legend":"\u003cp\u003epH study for Au-NGSPE in 0.1 M acetate buffer and 20 µM of tenofovir\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/0c85fb09fc6a804c5828b11d.png"},{"id":98780254,"identity":"2f2a2d6a-e8e4-492f-a07d-00d0cb2edb38","added_by":"auto","created_at":"2025-12-22 12:31:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13991,"visible":true,"origin":"","legend":"\u003cp\u003eDPV voltammogram of unmodified SPE and Au-NGSPE in 0.1 M acetate buffer and 20 mM Tenofovir\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/7102d4b9e10607e6a627d5cd.png"},{"id":98758586,"identity":"894ef579-7fe7-4324-9330-f540018d13f2","added_by":"auto","created_at":"2025-12-22 09:40:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":47977,"visible":true,"origin":"","legend":"\u003cp\u003e(A) DPV voltammograms for different concentrations of tenofovir in 0.1 M acetate buffer (B) Plot of calibration curve\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/a370eb52d44778c148ffe3aa.png"},{"id":98785458,"identity":"dba681da-92e2-444e-80dc-e2ff2a96593e","added_by":"auto","created_at":"2025-12-22 12:43:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1091539,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/50d4c6c3-75f3-471f-8ccb-bfbece8a027b.pdf"},{"id":98758556,"identity":"63ad4212-ae9f-4a86-8e03-9bf70a7608b5","added_by":"auto","created_at":"2025-12-22 09:40:26","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":50365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eScheme 1. Schematic illustration of the possible reaction mechanism of citrate capped AuNP and Tenofovir\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8253957/v1/93db22c62e4bbda60d41d5f9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a Silver Screen-Printed Paper Card Modified Gold Nitrogen Doped Graphene Electrode for Voltammetric Determination of Tenofovir","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTenofovir is a widely utilized antiretroviral medication classified as a nucleotide reverse transcriptase inhibitor (NRTI), commonly prescribed for the treatment of HIV [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and chronic hepatitis B infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, it is crucial to monitor its levels in the human body and the environment due to the potential risk of kidney and liver complications [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consequently, developing advanced, cost-effective, and precise sensing systems for determining tenofovir concentrations in real samples is essential [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Given its extensive clinical application and the risk of environmental contamination from pharmaceutical waste, accurate and sensitive methods for detecting and quantifying tenofovir in both pharmaceutical and ecological samples are vital [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe effectiveness of tenofovir disoproxil fumarate in managing HIV infections underscores the importance of quality control monitoring for this drug in its formulation and dissolution. Recently, tenofovir has been detected in bulk samples, pharmaceutical preparations, and tablet dosage forms utilizing a screen-printed carbon electrode. which serves as the first solid contact polymeric potentiometric sensor embedded with calix[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]arene as a supramolecular ionophore. The incorporation of a graphene nanocomposite layer significantly enhanced the sensor's performance and stabilized the potential signal compared to a control sensor, achieving a limit of detection (LOD) of 7.3 \u0026micro;M [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, a low-cost point-of-care assay was developed to assess tenofovir levels in urine accurately. Designed to support PrEP (pre-exposure prophylaxis) counseling, this urine tenofovir assay has shown improvements in both short-term and long-term adherence metrics, thus holding promise for enhancing long-term PrEP adherence [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGraphene, a remarkable two-dimensional nanocarbon material, inspires global interest for its potential to transform applications such as solar cells, fuel cells, supercapacitors, and sensors [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Graphene possesses several unique qualities, making it the best substrate for dispersing noble metal nanoparticles (NPs). These remarkable qualities, superb chemical and thermal stability, a vast surface area, and exceptional electrical conductivity, empower us to achieve extraordinary things [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Doping graphene with foreign atoms, like nitrogen, could improve its electrochemical performance and reactivity. Furthermore, compared to undoped graphene, incorporating nitrogen atoms into the lattice increases the active sites, creating a harmonious environment for a more even distribution of smaller metal nanoparticles [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Noble metal nanoparticles such as Au, Ag, and Pt were incorporated onto NG to develop new nanocomposites with enhanced electrochemical, electrocatalytic, and stability properties. There is a strong binding force between AuNPs and NG because of their high active surface area and superior catalytic capabilities [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVoltammetric techniques, particularly electroanalytical methods such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV), have gained attention as powerful tools for drug analysis [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The voltammetric technique enables the qualitative and quantitative determination of analytes at low concentrations with high specificity. These characteristics make the voltammetric technique suitable for drug confirmation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Electrochemical methods are instrumental in taking simultaneous measurements of multiple substances at once, and voltammograms can be displayed for different substances simultaneously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These techniques also offer high sensitivity, selectivity, low detection limits, and cost-effectiveness, making them suitable for determining tenofovir in complex matrices [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The ability of voltammetry to detect electroactive species based on their redox behavior allows for the precise analysis of tenofovir without the need for extensive sample preparation [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated the ability to detect tenofovir through electrochemical methods [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Some electrochemical sensors, developed with gold or carbon-based nanomaterials [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], exhibit extremely high sensitivity, swift detection, and can be employed as field-deployable point-of-care (POC) devices [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Among the different fabrication techniques used for drug detection, certain methods necessitate the incorporation of valves into rigid substrates such as glass and silicon, which involves many steps in both production and operation. This challenge can be addressed by using a paper substrate, which has proven to be an effective material in voltammetry, as it is currently the most cost-effective option and requires fewer steps in fabrication and operation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This article investigates the application of a paper card substrate in creating a screen-printed electrode for the differential pulse voltammetric detection of tenofovir, examining the electrochemical behavior toward the drug and various experimental parameters that affect its detection.\u003c/p\u003e "},{"header":"Experimental","content":"\u003ch2\u003eMaterials\u003c/h2\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eAll chemicals, materials, and reagents used in this study were of analytical grade. Nitrogen-doped graphene, chloroauric acid (HAuCl4), and silver conductive ink were sourced from Sigma Aldrich (USA). Sodium acetate and acetic acid were obtained from Kermel Chemicals (China). Potassium ferricyanide and potassium chloride were purchased from Harris Reagent (England), and KCl.Ag was acquired from Radiometer Analytical (France). HmbG Chemicals supplied Lead (II) chloride, L (+)-ascorbic acid, and urea. Uric acid, hydrochloric acid (HCl), and hydrazine hydrate were obtained from Sigma Aldrich (Germany). Sodium chloride, glucose, and citric acid were sourced from R\u0026amp;M Chemicals (UK), and phosphate buffer was obtained from Bendosen Laboratory Chemicals (Malaysia). Pure tenofovir powder was provided by Aminu Kano Teaching Hospital (AKTH), and the metallic gold card used in this research was also of analytical grade.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental Measurement\u003c/h3\u003e\n\u003cp\u003eThe morphology and dimensions of NG and Au-NG were characterized through Transmission Electron Microscopy (TEM) employing a JEOL JEM-2100F instrument. To evaluate the elemental composition and spectral characteristics of NG and Au-NG, Energy Dispersive X-ray Spectroscopy (EDX) was performed using a JEOL JSM 7600F Field Emission Scanning Electron Microscopy (FESEM) equipped with EDX capabilities. Additionally, electrochemical measurements were executed with an Autolab potentiostat operated via Nova software version 2.1.4. The potentiostat was coupled to a computer and the gold nitrogen-doped graphene screen-printed paper electrode (Au-NGSPE). CV of potassium ferricyanide was obtained by scanning potential from \u0026minus;\u0026thinsp;0.2 to 0.9 V at 50 mVs\u003csup\u003e-1\u003c/sup\u003e, while DPV for the study of tenofovir was scanned from 0.0 to 0.5 V at a scan rate of 100 mVs\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eSynthesis of Gold Nitrogen Doped Graphene (Au-NG)\u003c/h3\u003e\n\u003cp\u003eA 2 mL suspension of NG (concentration: 10 mg/mL) was mixed with 20 mL of a 0.05 M aqueous solution of HAuCl\u003csub\u003e4\u003c/sub\u003e. Sodium hydroxide (0.1 mol/L) was then added to adjust the pH to 8. After heating the mixture to 80\u0026deg;C for 35 minutes under constant stirring, 0.01 mL of hydrazine monohydrate (NH\u003csub\u003e2\u003c/sub\u003eNH\u003csub\u003e2\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO) was introduced. To facilitate the decoration of the NG nano sheets with gold, the mixture was allowed to react for an additional 30 minutes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDevice Design\u003c/h3\u003e\n\u003cp\u003eA three-electrode layer was screen printed with silver ink onto a paper card, then cured for 30 minutes at 120\u0026deg;C [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Following this, individual electrodes were cut out as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and stored in a sealed bag. The Au-NGSPE was assembled by drop-coating the surface of the working electrode with 20 \u0026micro;L of Au-NG, and allowing the solution to dry at room temperature before use [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscopy\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows the image of NG displaying the sheet like structure of graphene, while Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB illustrates the sheet like structure of the NG with Au nanoparticles in the size range of 70 nm to 80 nm decorated on it. The number of Au nanoparticles appears limited, likely due to the synthesis method and the ratio of NG to Au used in the process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnergy Dispersive X-Ray (EDX) Spectroscopy\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA presents the spectral image of NG, where the peaks of carbon, nitrogen, and oxygen are clearly visible, accompanied by their percentage weights. The sharp peak indicates that carbon constitutes the highest amount at 82.59% in NG. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB shows the spectral image of Au-NG, revealing peaks for carbon, oxygen, gold, and silver, along with their percentage weights. In this case, carbon still dominates at 68.8%, followed by gold at 20.05%. The absence of a nitrogen peak suggests a reaction between the nitrogen component and the gold. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC displays the spectral image of Au-NG in combination with tenofovir, where peaks for carbon, oxygen, gold, and silver are again observed, along with their percentage weights. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC exhibit notable differences in their elemental composition. In Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, the concentration of carbon is measured at 68.8%, alongside Au content of 20.05%. In contrast, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC shows a significant reduction in both carbon and gold percentages. Specifically, the amount of carbon is lower. However, the most striking change is observed in the oxygen content, which has dramatically increased from 8.47% in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB to an impressive 38.53% in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. This increase in oxygen suggests a substantial alteration in the chemical makeup or the environmental conditions impacting the samples represented by these Figures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eElectrochemical Response\u003c/h3\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, CV in 2 mM ferricyanide and 0.1 M KCl gave an anodic and cathodic peaks for the reduction and oxidation of ferricyanide [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e3\u0026minus;\u003c/sup\u003e to ferrocyanide [Fe(CN)\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e4\u0026minus;\u003c/sup\u003e. The unmodified SPE shows no definite reduction and oxidation peaks, while the Au-NGSPE showed a reduction peak around 0.4 V and an oxidation peak around 0.2 to 0.3 V, which agrees with the literature [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pH\u003c/h2\u003e \u003cp\u003eThe pH gives information about the redox behavior of the detection such as, sensitivity. When increase in pH is proportional to increase in current, it means the component is better oxidized or reduced at basic pH. From the pH study in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, tenofovir is better oxidized at basic pH than acidic pH with Au-NGSPE. The increase in peak current is due to deprotonation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The pH rises from pH 4 to 6, with a sharp decrease at pH 7 but gradually increases up to pH 10. The electro-oxidation of tenofovir is high at a basic pH. Therefore, pH 8 was chosen as the optimum pH for the detection of both tenofovir as the real sample analysis will be tested in urine sample, and both body pH and urine pH fall between the range of 7 to 8.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDifferential Pulse Voltammetry\u003c/h2\u003e \u003cp\u003eThe detection sensitivity was studied under DPV scan for both the unmodified electrode (UMSPE) and the Au-NGSPE in acetate buffer containing tenofovir. The Au-NGSPE exhibited a significant enhancement in the current measurements, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Therefore, different concentrations of tenofovir ranging from 20 \u0026micro;M to 100 \u0026micro;M were investigated using DPV in 0.1 M acetate buffer at pH 8 and a scan rate of 100 mV/s. The results produced a calibration plot with an excellent linear regression value (R\u0026sup2; = 0.9909) for the Au-NGSPE as displaced in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The detection of tenofovir using Au-NGSPE achieved a limit of detection (LOD) of 16.99 \u0026micro;M.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eValidation and Real (Urine) Sample Application\u003c/h2\u003e \u003cp\u003eThe proposed method\u0026rsquo;s applicability was evaluated through the analysis of tenofovir in urine samples. The results for both the original samples and those spiked with tenofovir were validated using gas chromatography-mass spectrometry (GC-MS) analysis. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrates a strong correlation between the newly developed analytical method and the established standard method, indicating that the proposed approach is suitable for tenofovir determination.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetermination of Tenofovir in urine sample\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSensor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTenofovir Added\u003c/p\u003e \u003cp\u003e(\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTenofovir Found (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% Recovery\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAu-NG/SPE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTenofovir\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (LOD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGC-MS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results indicated that the application of Au-NG significantly enhanced the sensitivity of the screen-printed electrode (SPE). This improvement is attributed to the synergistic effect between gold nanoparticles (AuNPs) and reduced graphene oxide (NG) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, the deposition of gold on the surface of NG increases charge transfer, leading to greater electrocatalytic activity and facilitating the electrochemical oxidation of tenofovir on the Au-NG/SPE surface. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Tenofovir can interact with gold nanoparticles on the surface of nitrogen-doped graphene through coordination bonding. The nitrogen-doped graphene can serve as a catalyst for the oxidation of tenofovir, leading to the formation of reactive oxygen species. Tenofovir can attach onto the surface of AuNPs through the amino group by electrostatic interaction and through the reactive oxygen species to give the oxidized tenofovir. As such, the more stable amine-reactive intermediates group of tenofovir can link with gold nanoparticles through the oxygen hybrid ring and hydroxyl group [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] as illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Both EDX and TEM analyses confirm the attachment of gold to the surface of NG; however, the amount of gold present is low compared to NG. A study investigating the variation of NG and Au was not conducted. There is potential to achieve significantly greater sensitivity by performing this study and by modifying the synthesis method, such as by introducing a different reducing agent. The calibration produced a strong linear plot. However, significant potential variations were observed when the linear range was expanded beyond the current limits. This resulted in high absorption on the electrode surface, which led to electrode damage and sample contamination. Foreign ions were selected based on those typically found in urine. The study found that these foreign ions did not significantly impact the detection of tenofovir in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The highest levels of interference were observed with glucose and urea. Ascorbic acid showed slight interference, while Na+, and K\u0026thinsp;+\u0026thinsp;had negligible effects, and uric acid demonstrated no interference at all. This suggests that all the ions have a broader potential range in conjunction with tenofovir, but the interference potential of glucose and urea may be more similar to that of tenofovir.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInterference Study for 100\u0026micro;m Tenofovir in the Presence of Foreign Ions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForeign ions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConc(\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePercentage of Interference (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUric Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe Au-NG/SPE was fabricated and applied for voltammetric detection of tenofovir. To the best of our knowledge, this is the first reported use of this approach for tenofovir detection. The results demonstrate that the synthesis method effectively allows for the attachment of gold (Au) onto the surface of the nanographene (NG). Additionally, modifying the screen-printed electrode (SPE) with Au-NG increased the sensitivity of the detection system. Calibration studies conducted for tenofovir demonstrated a robust linear response. The limit of detection was determined to be approximately 16 \u0026micro;M. Overall, the sensor shows promising applicability in the field.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate declaration\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003cem\u003e:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eNot Applicable\u0026apos; as no human study is involved\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution declaration\u003c/strong\u003e: M.L. and S.H. conceived and designed the study. M.L., A.A.A. and I.T. carried out the experimental work and optimization of the modified gold nitrogen-doped graphene electrode. S.H. performed the electrochemical measurements and analytical validation. M.L., A.B.S and U.S.A. analyzed the data. M.L., S.H. and A.B.S prepared Figures 1\u0026ndash;3. M.L. wrote the main manuscript text, and all authors contributed to editing and refining the final draft. All authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e. The research was supported by TETFUND National Research Fund; TETFund/DR\u0026amp;D/CE/NRF/CC/15/Vol1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMohareb AM, Miailhes P, Bottero J, Lascoux-combe C, Chas J, Maylin S, Gabassi A, Rougier H, Hyle EP, Delaugerre C, Lacombe K, Boyd A. Virological and serological outcomes in people with HIV-HBV coinfection who had discontinued tenofovir-containing antiretroviral therapy: Results from a prospective cohort study. J Virus Eradication. 2025;10(4):100574. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jve.2024.100574\u003c/span\u003e\u003cspan address=\"10.1016/j.jve.2024.100574\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuangrithi D, Silathong K. (2025). Factors predicting renal impairment associated with Tenofovir Disoproxil fumarate: Focus on patients with HIV and pre-existing metabolic abnormalities under the Thai National Health Security Scheme. 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J Phys Chem C. 2021;125(9):5006\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jpcc.0c08356\u003c/span\u003e\u003cspan address=\"10.1021/acs.jpcc.0c08356\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"gold nitrogen-doped graphene, tenofovir, paper card, screen-printed silver electrode, voltammetry","lastPublishedDoi":"10.21203/rs.3.rs-8253957/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8253957/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA new method for detecting tenofovir has been developed using a silver screen-printed paper card and voltammetric techniques. Gold nanoparticles (Au) were deposited onto nitrogen-doped graphene (NG) using a simple reduction method, resulting in a gold-decorated nitrogen-doped graphene (Au-NG) composite. The paper card was screen printed with silver ink, after which the composite was used to modify the paper card-based silver electrode and applied in the voltammetric detection of tenofovir. The sensor's characteristics were analysed using transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX). Electrochemical studies were performed using cyclic voltammetry (CV) and Differential pulse voltammetry (DPV). DPV with the modified electrode demonstrated a sensitivity 1.6 times higher than that of the unmodified electrode, achieving a linear calibration at a scan rate of 100 mV/s and pH 8 in acetate buffer. The sensor displayed strong resistance to interference from other ions and was successfully tested on real samples.\u003c/p\u003e","manuscriptTitle":"Development of a Silver Screen-Printed Paper Card Modified Gold Nitrogen Doped Graphene Electrode for Voltammetric Determination of Tenofovir","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 09:39:23","doi":"10.21203/rs.3.rs-8253957/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-02T12:16:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"48001565381235635695316991684003248472","date":"2026-02-01T09:57:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-30T16:26:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188529858283204765204193210577836957630","date":"2026-01-29T17:05:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52803466219503819601005477440062533592","date":"2026-01-29T12:10:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-29T10:59:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247298361506269564465442236134339194036","date":"2025-12-21T10:24:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-19T13:39:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18109025029858606514401254646525530761","date":"2025-12-16T19:24:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-16T09:36:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-11T05:03:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-10T20:50:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2025-12-10T20:46:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30d47e19-0892-45f9-862c-13cf6b77fe1d","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T15:24:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 09:39:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8253957","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8253957","identity":"rs-8253957","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}