Gold Nanostructure-Enhanced Immunosensing: Ultra-Sensitive Detection of VEGF Tumor Marker for Early Disease Diagnosis

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Sajedi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3881185/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2024 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract We present an advanced electrochemical immunosensor designed for precise detection of the Vascular Endothelial Growth Factor (VEGF). The sensor is constructed on a modified porous gold electrode through a fabrication process involving the deposition of silver and gold on an FTO substrate. Employing thermal annealing and a de-alloying process, the silver is eliminated from the electrode, resulting in a reproducible porous gold substrate. Utilizing a well-defined protocol, we immobilize the heavy-chain (VHH) antibody against VEGF on the gold substrate, facilitating VEGF detection through various electrochemical methods. Remarkably, this immunosensor achieves an outstanding performance, featuring an impressive detection limit of 0.1pg/ml and an extensive linear range from 0.1pg/ml to 0.1µg/ml. This emphasizes its exceptional ability to precisely measure biomarkers across a wide concentration spectrum. The robust fabrication methodology employed in this research underscores its potential for widespread application, offering enhanced precision, reproducibility, and remarkable detection capabilities for the developed immunosensor. Biological sciences/Biotechnology/Nanobiotechnology Physical sciences/Nanoscience and technology/Nanoscale devices Electrochemical immunosensor VEGF detection Porous gold electrode Fabrication methodology Biomarker measurement Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Today, early diagnosis and prompt treatment of various cancers are very important, where delayed diagnosis directly leads to higher mortality rates and lower treatment costs 1 , 2 . According to WHO, tumors are responsible for 90% of human cancer cases and can emerge in various body regions, including the lungs, breasts, bladder, prostate, intestines, and kidneys. Vascular endothelial growth factor (VEGF) is recognized as a significant cancer biomarker, playing a crucial role in the formation of cancer tumors 3 . Monitoring VEGF levels enables the estimation of tumor status in many cases. Normally, the cancer threshold limit for the VEGF biomarker is 207 pg/ml in serum and 23 pg/ml in the blood plasma 3 . Of course, the VEGF threshold value may slightly change in various cancer types 3 . In fact, increased levels of the VEGF biomarker have been observed in various tumor-associated cancers, including carcinoma, lung, colon prostate, brain, kidney, and breast cancers 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . Consequently, determining VEGF levels in clinical samples is a normal way for diagnosis, prognosis, and therapeutic monitoring of various cancers. The detection of VEGF biomarkers can be achieved using different biosensor transducers like optical 13 , fluorescence 14 , field-effect transistor 15 , quartz crystal microbalance 16 , and surface plasmon resonance 17 . On the other hand, electrochemical immunosensors offer a highly sensitive and specific approach to detecting various antibody/antigen elements 18 . Electrochemical immunosensing of VEGF typically involves immobilizing VEGF-specific antibodies onto a transducer surface, which interacts with VEGF molecules in a sample, leading to a measurable electrochemical response 19 . This approach offers several advantages, such as rapid detection, minimal sample requirement, and the ability to detect VEGF in complex biological media 20 . Generally, incorporating nanostructures into the electrode design of electrochemical biosensors have been shown to enhance the effective electroactive surface area, charge transfer, and thus the sensitivity of the electrodes 21 , 15 . Various nanomaterials, including gold 22 , silver 23 , graphene 24 , and nanocomposites 25 , have been employed in the design of the electrode surface. Among them, Au nanostructures are the preferred choice due to their high conductivity, biocompatibility, and its resistance to rapid oxidation. Gold nanostructures can be deposited onto the surface using various methods such as physical vapor deposition (PVD), chemical vapor deposition (CVD), hydrothermal synthesis, and reducing agent synthesis 22 , 23 , 24 , 25 . In the current research, the main goal is to design and fabricate an Au nanostructured electrode for ultra-Sensitive immounosensing detection of VEGF through various electrochemical methods. The nonporous Au electrode has been fabricated using the simple and reliable dealloying method. Initially, thin films of silver and gold were deposited onto the Fluorine-doped tin oxide (FTO) substrate using PVD and then the surface was modified through an annealing procedure. The dealloying method with HNO 3 solution effectively removes the silver from the structure to obtain a nonporous Au electrode. The resulting electrode serves as the working electrode and offers the advantages of biocompatibility and high reproducibility. By implementing a straightforward procedure for antibody immobilization on the gold substrate, VEGF detection was accomplished through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Finally, measurement of the VEGF antigen using the designed immunosensor has been carried out, and the sensor demonstrates a high linear detection range with a very low detection limit of 0.1 pg/ml. 2. Experimental section 2.1. Chemicals and materials For the PVD process, high-purity Au and Ag metallic pellets were used. These metals were deposited onto the FTO substrates (15 Ωsq − 1), which were cut into pieces of 0.8×1.25 cm 2 with a thickness of 2 mm. In the experiments, phosphate-buffered saline (PBS) was used as a buffer solution with a pH of approximately 7.4. Additional chemicals used included mercaptoacetic acid (MAA), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-Hydroxysuccinimide (NHS), all of which were obtained from Sigma–Aldrich. Also, gelatin, variable domain of heavy-chain antibody (VHH) against VEGF 26 , and VEGF antigens 27 , ethanol (99.9% purity) were also utilized in the study. For the electrochemical measurements, following chemicals were used: potassium chloride (KCl), potassium ferricyanide (K 3 [Fe (CN) 6 ]), potassium ferrocyanide (K 4 [Fe (CN) 6 ]), and deionized water (DI water). These chemicals were utilized as received without further purification beyond their initial specifications. 2.2. Instruments The morphology of the fabricated electrodes was characterized by field emission scanning electron microscopy (FE-SEM) equipped with EDS (FEI Nova NanoSEM-450 model). X-ray diffraction (XRD) was used for phase crystalline identification (X’Pert Pro MPD system equipped with Cu-Kα radiation). The wetting properties during the surface modification were studied by a contact angle measurement device (Jikan, Iran). The cyclic voltammetry (CV), electrochemical impedance measurement (EIS), and differential pulse voltammetry (DPV) were performed with the Origalysis potentiostat system (ElectroChem SAS, France) through a three electrodes electrochemical cell containing Ag/AgCl as a reference electrode, Au plate as a counter electrode and the nano-porous gold electrode as the work electrode. 2.3. Biosensors Fabrications We prepared the primary substrate before the physical vapor deposition by following a specific washing protocol. The surface of the FTO electrodes was scrubbed with acetone, ethanol, and deionized water. Subsequently, we ultrasonicated them for 15 minutes and then dried them inside an oven. After that, different thicknesses of the silver film were deposited on the FTO electrodes, followed by the deposition of a 5 nm gold thin film, all by the PVD method. The pressure in the PVD chamber was maintained at 1×10 − 6 Torr before the deposition started. Also, the growth rate for deposition was adjusted to 0.1 nm/s, and thin film thickness was controlled by quartz crystal microbalance. To fabricate an Ag-Au alloy nanostructure, we subjected the electrodes with an Ag-Au thin film to a thermal annealing process in a furnace for 2 hours at the temperature of 550 o C. Afterward, to modify the electrode, the annealed electrode was dipped into 65% nitric acid at room temperature for 15 minutes to induce dealloying and remove the Ag material from the electrode. Finally, the modified electrode was thoroughly rinsed with DI water. To biofunctionalize the gold surface with antibody, we followed a series of steps, including, first 50 µL of an MAA solution (14 mmol/L) was applied to the working electrode at room temperature for 2 hours. Then, the electrode was washed with ethanol to remove any unbound MAA from the surface. To activate the carboxylic groups, a solution of 50 µL of 50 mM EDC/NHS (1:1) in PBS buffer at pH 6.0 put on the working electrode surface for one hour at room temperature. Next, 50 µL of 10 µg/ml VHH anti-VEGF was deposited on the activated electrode overnighted at 4 ℃ for antibody fixation. To prevent and block non-specific bindings, we applied a gelatin solution (50 µL of 20 mg/mL in PBS) to the electrode for 45 minutes 28 . Subsequently, different VEGF concentrations were introduced to the activated surface for 45 minutes at 4 ℃ to characterize the biosensor. Throughout all procedures, the electrode was kept in a dark ambient environment. After activating the carboxylic groups, any unbound reagents were washed away with a PBS solution (pH 7.4) at each stage. For a visual representation of the fabrication of the sensor, the antibody fixation and antigen detection stages are all shown in Fig. 1 . 3. Result and discussion 3.1. Characterization of the electrode surface Figure 2 illustrates the different fabricated electrodes through alloying and dealloying processes. Figure 2 a shows the morphologies of the alloying structure of Ag-Au on the FTO substrate. Due to the thermal annealing process, semi-spherical Ag-Au nanoparticles are shaped on the FTO surface, wherein a hybrid layer containing a 25 nm silver thin film and a 5 nm gold thin film were initially deposited using the PVD system. In Fig. 2 b, the Au nano-porous structures are visible after the dealloying process. The energy-dispersive X-ray spectroscopy (EDS) confirms the complete removal of silver during the dealloying process, as shown in Fig. 2 c. Figures 2 d and 2 e show the difference in hydrophilicity of the optimized Ag-Au electrode before and after the dealloying process, as demonstrated via the contact angle measurement. The porosity of the formed gold nanostructures directly effects on the measured contact angle, as the presence of nanopores on the surface increases the hydrophilicity of the working electrode. After dealloying, the contact angle decreased from 21.1 \(^\circ\) to 10.4 \(^\circ\) , indicating that the surface had become more hydrophilic. This almost 50% decrease in the contact angle demonstrates improved hydrophilicity of the formed structure. Consequently, the low contact angle of the electrode surface leads to better connecting biomolecules to the electrode, enhancing the biofunctionality and detection capability of the sensor. To confirm the phases and components of the optimized electrode, XRD patterns were conducted, and the results are shown in Fig .2f. In addition, the XRD patterns of the bare FTO electrode, the bi-layer of 25 nm Ag and 5 nm Au electrode annealed at 550 o C, and the final nano-porous gold nanostructures have been shown for comparison purposes. The XRD patterns provide evidence for the creation Ag-Au nanoparticles, and then the porous gold particles on the FTO substrate. The peaks of Au, Ag and FTO have many similar diffraction angles leading to appearance of several broadened peaks in the XRD spectrum. The crystallographic directions of various planes have also been displayed in the spectrums. The primary crystallographic orientations for the FTO substrate correspond to the (110), (101), (200), (211), (310), and (301) planes, with corresponding 2 \({\theta }\) values of 26.6 \(^\circ\) , 33.8 \(^\circ\) , 37.8 \(^\circ\) , 52 \(^\circ\) , and 62 \(^\circ\) , respectively. We observe even stronger peaks for the gold and silver nanoparticles. The prominent peaks of Au-Ag nanoparticles are observed at 2 \({\theta }\) values of 38.2 \(^\circ\) , 64.6 \(^\circ\) , and 77.6 \(^\circ\) with the miller indices (hkl) of (111), (220), and (311) for the primary crystallographic orientations. After dealloying, nano-porous gold nanoparticles remain with the plane orientations of (111) and (220) at 2 \({\theta }\) the angles of 37.5 \(^\circ\) and 43.6 \(^\circ\) , respectively. There are two important parameters in forming of porous nanostructures during the alloying and dealloying processes: the ratios of gold to silver in the PVD process and the annealing temperature in the thermal processing. Both of these parameters determine the size and porosity of the final porous nanostructures. Figure 3 shows the FE-SEM images of fabricated gold nanostructures with different thickness ratio of Ag and Au layers after the dealloying process, at the same annealing temperature of 550 o C. The figure shows that the thickness ratio of silver to gold considerably affects the size of the dealloyed nanostructures. No observable porosity was revealed after post-dealloying in the case of 5 nm silver and 5 nm gold in Fig. 3 a. Conversely, as the silver thicknesses increase to 10, 15, 20, 25, and 30 nm, a corresponding boost in the porosity of the emerged post-dealloying structure, is revealed. Nonetheless, no noticeable alteration occurs for silver dimensions of 25 nm and 30 nm. This conducts us to select the case of 25 nm silver and 5 nm gold arrangement as the best initial Ag-Au thicknesses for the alloy-dealloy process. In continuation of Fig. 3 results, the morphological structures of various arrangements involving 10 nm gold thin film integrated with different silver thicknesses of 5, 20, and 40 nm were investigated, as shown in Fig. 3 (g-i). Again, no porosity was exhibited after dealloying in the case of the 10 nm gold thin film coupled with the 5 nm silver thin film. As observed previously, raised silver content correlates with increased porosity. Drawing from these figures’ analysis, one can infer that the thickness of initial silver substrate compared to the finial gold over-layer plays a crucial role in initiating and boosting the porosity of the structure. Simultaneously, the amount of gold dictates the ultimate dimensions of the formed nanostructure. However, the use of more than 10 nm gold thickness was generally limited due to the increased gold consumption, which posed economic inefficiency concerns. To investigate and obtain an optimized annealing temperature for the thermal process, the morphologies and the average sizes of nanostructures after the dealloying process were measured as a function of the annealing temperature. As the temperature increases, the porosities become more apparent, and their surface distribution increases in the final structure. Despite this issue, according to the size distribution histograms, the overall size structure is similar for both temperatures of 550 and 600 o C. Therefore, the electrode with the annealing temperature of 550 o C with thicknesses of 25 nm silver and 5 nm gold was selected as the optimized electrode for further sensing investigations. In addition, further results of this investigation have been shown in Fig. S1 , in the supplementary information. 3.2 Electrochemical characterization of the fabricated electrodes In this section, the modified electrodes were subjected to electrochemical characterization via cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The results for the bare FTO, the optimized Au-Ag electrode, and the optimized Au-Ag electrode after the dealloying process, were compared in Fig. 4 a and b. Initially, the peak-to-peak potential of the FTO substrate was measured at 624 mV, and the maximum current was 620 µA. According to the EIS result, the FTO surface resistance was about 194 Ω. After modifying the FTO surface to the optimized Ag-Au layer (before the dealloying process), the silver peaks appear in the CV result at the potentials of 141 mV and 552 mV. In this case, the surface resistance reaches 56 Ω, indicating about 72% decrement relative to that of the bare FTO electrode. In the next step, silver was dealloyed and the porous Au nanoparticles were produced. As shown in Fig. 4 a and b, the current peak value of the modified porous gold electrode reaches 830 µA, indicating about 34% increase in the current compared to the pure FTO substrate. Although the peak current of the Ag-dealloyed electrode is about 12% less than this electrode before dealloying, the peak-to-peak potential for the dealloyed electrode is about 35% less than this electrode before the dealloying process. This indicates better reversibility of the dealloyed electrode against redox reactions relative to the same case before dealloying. In addition, the dealloyed electrode has a 235 mV peak-to-peak potential which is about 33% less than that of the bare FTO. Finally, the resistance of the modified dealloyed surface relative to the FTO surface decreased by 93%, reaching about 14Ω. In addition, the stability of the electrode was investigated in the first, 20th, 50th, and 100th cycles, and it is shown in Fig. 4 c that no significant change was seen after the 50th cycle. In order to have an insight into the time of the dealloying process, we measured the open circuit potential (OCP) to evaluate the impact of nitric acid dealloying on the electrode surface. As illustrated in Fig. 4 d, after approximately 10 minutes, nearly 94% of the change of OCP has taken place. When the OCP stabilizes, it indicates that the dealloying process is fully developed and all the silver has been completely removed. In our experiments, we have chosen an alloying time of approximately 15 minutes to ensure the complete removal of silver from the electrode surface. In Fig. S2, the influence of the annealing temperature on the electrochemical response of the modified electrode at three annealing temperatures of 450, 550, and 600 ℃ has been investigated. The diameter of the semicircle in the Nyquist plot represents the charge transfer resistance ( \({\text{R}}_{\text{c}\text{h}}\) ). The \({\text{R}}_{\text{c}\text{h}}\) at the temperatures of 450 and 600 ℃ is higher than that of 550 ℃. The same scenario is also repeated for the peak current of the 550 ℃ sample, in the CV results. As depicted in Fig. S3, it is apparent that the square root of the scan rate shows a clear linear relationship with the anodic peak current. This observation strongly indicates that the redox reaction involving the nano-porous gold nanostructures follow a diffusion-controlled process. 3.3 Immunosensing characterization and analytical performance As previously mentioned in Fig. 1 , the process of anchoring antibodies to the modified electrode and detecting the VEGF biomarker involves using MAA, EDC, and NHS. These compounds are utilized for immobilization on the modified electrode, facilitating the covalent attachment of antibodies to the transducer element. As shown in Fig. 5 , the immunosensensing characterization has been performed by CV, EIS, and DPV measurements. At first, the CV, EIS, and DPV tests of the bare dealloyed electrode. we prepared these tests after immobilizing the antibody on the surface. The predominant covalent bonding of the VHH to the electrode surface leads to a further reduction in the anodic peak current to 717 \({\mu }\text{A}\) . After that, to block the empty places on the surface, we used gelatin, appearing in the increment of the electrode resistance from (56 Ω) for the antibody state to (107 Ω) after the block stage. In this case, the reduction of the current peak reads from (717 µA) to (610 µA). In the last step, VEGF antigen was placed on the surface to ensure the correct connection of all steps. As it is clear in the figure, in the last stage, which is also the antigen detection stage, the current peak in the CV diagram has decreased to (525 µA) in addition to about 31% increment of the peak-to-peak voltage compared to the state of Au porous. The EIS graph shows the resistance increment from (107 Ω) to (204 Ω), and the DPV diagram also shows the decrease in the current, which are all proofs of antigen detection by the desired immunosensor. Also, result of the numerical values of the electrical parameters correspond to equivalent EIS circuit was used and the results of obtained parameters have been summarized in Fig. 5 d. Also, the equivalent circuit for modeling the EIS result has been shown in the inset of Fig. 5 b. Figure 6 shows the calibration curve result of the VEGF determination by the fabricated sensor. In each step, to find the linear range, the difference between the resistances ( \(\varDelta {\text{R}}_{\text{c}\text{t}}\) ) with the state where VEGF is not present in the solution is measured. The equation of concentrations ranges from 0.1 pg/ml to 0.1 µg/ml can be expressed as ∆R ct (Ω) = 84.35C Log (VEGF) [gr/ml] + 1146.6 with a regression coefficient of 0.97. This immunosensor has a very good limit of detection of 0.1 \(\) pg/ml. Also, in Fig. 6 c the selectivity of our immunosensor has been demonstrated. It shows that the interference species produce currents that are lower than 20% of the VEGF signal. This finding indicates the high-level selectivity of this sensor for detecting VEGF, as it suggests minimal interference from other substances. In Table.1, the characteristics of different biosensors for VEGF in the literature have been compared by this sensor. The findings illustrate that the nano-porous Au electrode outperforms various biosensors, especially regarding the limit of detection (LOD) and linear response range when of VEGF detection. Table.1: Comparison of the analytical capabilities of different immunosensors documented for the detection of VEGF with the current research. Biosensor substrate Detection method Linear range LOD Ref. Au/3-MPA/EDC-NHS/VEGF-R1 CV EIS 10–70 pg/ml 38 pg/ml 2 POLY (3,4-ethylene dioxythiophene) (PEDOT)/Gold Nanoparticle (Au NP) Composites EIS 1–20 pg/ml 0.5 pg/ml 29 PdPtMo CME NPs EIS 10–10 6 pg/ml 8.2 pg/ml 30 RGO/Au NPs/11MUA/Antibody/VEGF/HRP (Sandwich type) CV SWV EIS 2-20000 ng/ml 6 fg/ml 31 cellulose paper fluorescence control (FApt) 100–5000 ng/ml 137 ng/ml 32 Au nano porous/MAA/EDC-NHS/VHH/Gelatin/VEGF CV EIS \({10}^{-4}{-10}^{2}\) ng/ml 0.1 pg/ml This work 4. Conclusion In this study, we have successfully designed an electrochemical immunosensor for detecting the VEGF tumor marker based on a modified porous gold electrode. The electrode is created through a series of straightforward processes, involving silver and gold deposition on an FTO substrate, followed by thermal annealing and dealloying to entirely eliminate silver from the electrode surface. Employing a comprehensive protocol for antibody attachment to the gold substrate, we conducted VEGF detection using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Impressively, this immunosensor exhibits a remarkable detection limit of 0.1 pg/ml, making it an ideal tool for highly sensitive VEGF tests. Furthermore, the linear range extends from 0.1 pg/ml to 0.1 µg/ml, indicating not only a low limit of detection but also a wide dynamic range for accurate measurements. In conclusion, this research not only establishes a robust method for sensitive electrode fabrication but also highlights its suitability for widespread adoption, positioning it as a promising candidate for scaling up in various scientific and medical contexts. Declarations Author Contribution S.Y., H.S., R.H.S. and A.M. conceived of the presented idea and developed the theory and verified the analytical methods. S.Y., M.K. and H.S. carried out the experiments. 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Additional Declarations No competing interests reported. Supplementary Files SupportingInformationscientificreportnature.docx Cite Share Download PDF Status: Published Journal Publication published 07 May, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Feb, 2024 Reviews received at journal 08 Feb, 2024 Reviewers agreed at journal 29 Jan, 2024 Reviewers agreed at journal 29 Jan, 2024 Reviewers invited by journal 29 Jan, 2024 Editor assigned by journal 24 Jan, 2024 Editor invited by journal 24 Jan, 2024 Submission checks completed at journal 24 Jan, 2024 First submitted to journal 20 Jan, 2024 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. 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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-3881185","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":269209136,"identity":"c06b0941-260e-4503-ba26-fccf042b80d8","order_by":0,"name":"Sadaf Yarjoo","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Sadaf","middleName":"","lastName":"Yarjoo","suffix":""},{"id":269209137,"identity":"f1808c8e-22d6-412c-84b6-a4238e20103e","order_by":1,"name":"Hossein Siampour","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Hossein","middleName":"","lastName":"Siampour","suffix":""},{"id":269209138,"identity":"ea285858-de86-4631-82d4-8b48e1f9cd64","order_by":2,"name":"Mehrsa Khalilipour","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Mehrsa","middleName":"","lastName":"Khalilipour","suffix":""},{"id":269209139,"identity":"17e7399b-5ba5-4a05-8bf0-dadc5b7ee549","order_by":3,"name":"Reza H. Sajedi","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Reza","middleName":"H.","lastName":"Sajedi","suffix":""},{"id":269209140,"identity":"273c8134-2161-485d-ae4c-faa805972801","order_by":4,"name":"Hassan Bagheri","email":"","orcid":"","institution":"Baqiyatallah University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hassan","middleName":"","lastName":"Bagheri","suffix":""},{"id":269209141,"identity":"d2f5f44a-6577-4f50-afb1-1593432d7a0c","order_by":5,"name":"Ahmad Moshaii","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3PsQrCMBCA4RPBqeCaoNQnEFKEqG/TTl0UBEE6SEkRzOKrOIjgrATa5R6gbk6dXMRFQcS4iKBE3RzyT8fBx3EANtt/VgFgxNUDe6zW35DWrwQgEM/EWFPKYncadMK5nCyOURRDVa5LKjIQjtj2Zoz0V5gOKaICgj5s0ETyXoU4d5L3GE2m+oscYCOMJCzohZFQ29Y5ucbQ+Ex8XtNXfE04TUQZ2EeCyGt1Rjz9y6grUuV4GAgzyWRB95e4wbPJcivGsetmSh1M5CUHoPQTsNlsNtubbkBMT7JI6HPUAAAAAElFTkSuQmCC","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":true,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Moshaii","suffix":""}],"badges":[],"createdAt":"2024-01-20 09:47:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3881185/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3881185/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-60447-2","type":"published","date":"2024-05-07T04:00:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50308497,"identity":"48c80b74-833c-40eb-9d15-9a092f6384da","added_by":"auto","created_at":"2024-01-29 13:52:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":455146,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of preparation of the modified electrode and preparation steps of the immunosensing detection of VEGF.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3881185/v1/7c2b70a039bc585c7776e565.png"},{"id":50308498,"identity":"07c2c400-35f5-4466-a731-54c5a56f26aa","added_by":"auto","created_at":"2024-01-29 13:52:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":509434,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Morphology of spherical Ag-Au nanoparticles after the annealing process, (b) Morphology of the nano-porous Au structures after the dealloying process, (c) EDS quantitative result of Ag-Au nanoparticles before and after dealloying. After dealloying, all silver was removed. The contact angle measurement for the optimized electrode after the annealing process (d), with measured contact angle of 21.1\u003csup\u003eo\u003c/sup\u003e, and after the dealloying (e) with the measured 10.4\u003csup\u003eo\u003c/sup\u003e. The graph (f) shows XRD pattern of FTO, Ag-Au alloy and porous Au electrode.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3881185/v1/0d3b398883e0cf5b51f4741d.png"},{"id":50308501,"identity":"de90a9fe-11ef-4bce-afa6-5b52a50ef9a5","added_by":"auto","created_at":"2024-01-29 13:52:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1104812,"visible":true,"origin":"","legend":"\u003cp\u003eFE-SEM images of nano-porous gold with different gold and silver layer thicknesses with the same annealing temperature of 550 \u003csup\u003eo\u003c/sup\u003eC, including: a) 5 nm Ag@ 5 nm Au, b) 10 nm Ag@ 5 nm Au, c) 15 nm Ag@ 5 nm Au, d) 20 nm Ag@ 5 nm Au, e) 25 nm Ag@ 5 nm Au, f) 30 nm Ag@ 5 nm Au, g) 5 nm Ag@ 10 nm Au, h) 20 nm Ag@ 10 nm Au, i) 40 nm Ag@ 10 nm Au.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3881185/v1/873b383b61cdf864028a7bba.png"},{"id":50308499,"identity":"daddcc4a-57e3-4e4e-87b1-c4b693b0a0d2","added_by":"auto","created_at":"2024-01-29 13:52:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":357312,"visible":true,"origin":"","legend":"\u003cp\u003eelectrochemical of diagram of modification step a) CV chart, b) EIS chart, c) Investigating the stability of the modified electrode after the first, 20th, 50th, and 100th cycle and d) Open circuit potential of the Au-Ag nanoparticles over the first 1200 s of exposure to the HNO\u003csub\u003e3\u003c/sub\u003e solution.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-3881185/v1/7620a26fb5b349d47eb464ee.png"},{"id":50308502,"identity":"834f588f-056d-4eaf-94d8-740f1a2a96c9","added_by":"auto","created_at":"2024-01-29 13:52:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":315528,"visible":true,"origin":"","legend":"\u003cp\u003eVarious electrochemical techniques including a) CVs with a scan rate of 50 mV/s, b) Nyquist plot (EIS - Electrochemical Impedance Spectroscopy), c) DPV were employed in a solution containing 2.5 mM [Fe(CN)\u003csub\u003e6\u003c/sub\u003e] \u003csup\u003e-3/-4\u003c/sup\u003e to monitor the stepwise immobilization process of biological agents on the nano-porous particles modified electrode. The EIS plots from left to right corresponded to the stages of modification with nano-porous nanoparticles, immobilization of VHH, gelatin blocking, and VEGF molecule capture on the electrode. Additionally, consistent color coding was used across all figures for clarity and consistency. The concentration of VEGF in this study was 1 pg/mL, and d) Numerical values of the electrical equivalent circuit of immunosensor obtained.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-3881185/v1/acc971d360a37733e2013363.png"},{"id":50308500,"identity":"29cec078-c628-4138-8d58-c263d4ce53a9","added_by":"auto","created_at":"2024-01-29 13:52:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":205757,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The EIS response of VEGF concentrations ranging from 10\u003csup\u003e-4\u003c/sup\u003e-10\u003csup\u003e2\u003c/sup\u003e ng/ml, (b) calibration curve response was measured in a 2.5 mM [Fe (CN)\u003csub\u003e6\u003c/sub\u003e] \u003csup\u003e-3/-4 \u003c/sup\u003esolution and (c) characterization of different samples to determine selectivity.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-3881185/v1/1f6c2aeb4ffa3d2ed730f39b.png"},{"id":56140578,"identity":"3699f77d-be48-4f39-9740-75bb99f40878","added_by":"auto","created_at":"2024-05-09 04:36:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3280830,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3881185/v1/e11b8370-f5a7-4917-85c6-a6f009a21256.pdf"},{"id":50308503,"identity":"8d17d177-4601-4b5d-8064-9f6395335273","added_by":"auto","created_at":"2024-01-29 13:52:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1441523,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationscientificreportnature.docx","url":"https://assets-eu.researchsquare.com/files/rs-3881185/v1/ce8114034e6cb5fe688a60f9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gold Nanostructure-Enhanced Immunosensing: Ultra-Sensitive Detection of VEGF Tumor Marker for Early Disease Diagnosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eToday, early diagnosis and prompt treatment of various cancers are very important, where delayed diagnosis directly leads to higher mortality rates and lower treatment costs \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. According to WHO, tumors are responsible for 90% of human cancer cases and can emerge in various body regions, including the lungs, breasts, bladder, prostate, intestines, and kidneys. Vascular endothelial growth factor (VEGF) is recognized as a significant cancer biomarker, playing a crucial role in the formation of cancer tumors \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Monitoring VEGF levels enables the estimation of tumor status in many cases.\u003c/p\u003e \u003cp\u003eNormally, the cancer threshold limit for the VEGF biomarker is 207 pg/ml in serum and 23 pg/ml in the blood plasma \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Of course, the VEGF threshold value may slightly change in various cancer types \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In fact, increased levels of the VEGF biomarker have been observed in various tumor-associated cancers, including carcinoma, lung, colon prostate, brain, kidney, and breast cancers\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Consequently, determining VEGF levels in clinical samples is a normal way for diagnosis, prognosis, and therapeutic monitoring of various cancers.\u003c/p\u003e \u003cp\u003eThe detection of VEGF biomarkers can be achieved using different biosensor transducers like optical \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, fluorescence \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, field-effect transistor \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, quartz crystal microbalance \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and surface plasmon resonance \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. On the other hand, electrochemical immunosensors offer a highly sensitive and specific approach to detecting various antibody/antigen elements \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Electrochemical immunosensing of VEGF typically involves immobilizing VEGF-specific antibodies onto a transducer surface, which interacts with VEGF molecules in a sample, leading to a measurable electrochemical response \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This approach offers several advantages, such as rapid detection, minimal sample requirement, and the ability to detect VEGF in complex biological media \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGenerally, incorporating nanostructures into the electrode design of electrochemical biosensors have been shown to enhance the effective electroactive surface area, charge transfer, and thus the sensitivity of the electrodes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Various nanomaterials, including gold \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, silver \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, graphene \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and nanocomposites \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, have been employed in the design of the electrode surface. Among them, Au nanostructures are the preferred choice due to their high conductivity, biocompatibility, and its resistance to rapid oxidation. Gold nanostructures can be deposited onto the surface using various methods such as physical vapor deposition (PVD), chemical vapor deposition (CVD), hydrothermal synthesis, and reducing agent synthesis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn the current research, the main goal is to design and fabricate an Au nanostructured electrode for ultra-Sensitive immounosensing detection of VEGF through various electrochemical methods. The nonporous Au electrode has been fabricated using the simple and reliable dealloying method. Initially, thin films of silver and gold were deposited onto the Fluorine-doped tin oxide (FTO) substrate using PVD and then the surface was modified through an annealing procedure. The dealloying method with HNO\u003csub\u003e3\u003c/sub\u003e solution effectively removes the silver from the structure to obtain a nonporous Au electrode. The resulting electrode serves as the working electrode and offers the advantages of biocompatibility and high reproducibility. By implementing a straightforward procedure for antibody immobilization on the gold substrate, VEGF detection was accomplished through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Finally, measurement of the VEGF antigen using the designed immunosensor has been carried out, and the sensor demonstrates a high linear detection range with a very low detection limit of 0.1 pg/ml.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1. Chemicals and materials\u003c/h2\u003e\n\u003cp\u003eFor the PVD process, high-purity Au and Ag metallic pellets were used. These metals were deposited onto the FTO substrates (15 Ωsq\u0026thinsp;\u0026minus;\u0026thinsp;1), which were cut into pieces of 0.8\u0026times;1.25 cm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e with a thickness of 2 mm. In the experiments, phosphate-buffered saline (PBS) was used as a buffer solution with a pH of approximately 7.4. Additional chemicals used included mercaptoacetic acid (MAA), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-Hydroxysuccinimide (NHS), all of which were obtained from Sigma\u0026ndash;Aldrich. Also, gelatin, variable domain of heavy-chain antibody (VHH) against VEGF \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and VEGF antigens \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, ethanol (99.9% purity) were also utilized in the study.\u003c/p\u003e\n\u003cp\u003eFor the electrochemical measurements, following chemicals were used: potassium chloride (KCl), potassium ferricyanide (K\u003csub\u003e3\u003c/sub\u003e[Fe (CN)\u003csub\u003e6\u003c/sub\u003e]), potassium ferrocyanide (K\u003csub\u003e4\u003c/sub\u003e[Fe (CN)\u003csub\u003e6\u003c/sub\u003e]), and deionized water (DI water). These chemicals were utilized as received without further purification beyond their initial specifications.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2. Instruments\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eThe morphology of the fabricated electrodes was characterized by field emission scanning electron microscopy (FE-SEM) equipped with EDS (FEI Nova NanoSEM-450 model). X-ray diffraction (XRD) was used for phase crystalline identification (X\u0026rsquo;Pert Pro MPD system equipped with Cu-K\u0026alpha; radiation). The wetting properties during the surface modification were studied by a contact angle measurement device (Jikan, Iran). The cyclic voltammetry (CV), electrochemical impedance measurement (EIS), and differential pulse voltammetry (DPV) were performed with the Origalysis potentiostat system (ElectroChem SAS, France) through a three electrodes electrochemical cell containing Ag/AgCl as a reference electrode, Au plate as a counter electrode and the nano-porous gold electrode as the work electrode.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3. Biosensors Fabrications\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eWe prepared the primary substrate before the physical vapor deposition by following a specific washing protocol. The surface of the FTO electrodes was scrubbed with acetone, ethanol, and deionized water. Subsequently, we ultrasonicated them for 15 minutes and then dried them inside an oven. After that, different thicknesses of the silver film were deposited on the FTO electrodes, followed by the deposition of a 5 nm gold thin film, all by the PVD method. The pressure in the PVD chamber was maintained at 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e Torr before the deposition started. Also, the growth rate for deposition was adjusted to 0.1 nm/s, and thin film thickness was controlled by quartz crystal microbalance. To fabricate an Ag-Au alloy nanostructure, we subjected the electrodes with an Ag-Au thin film to a thermal annealing process in a furnace for 2 hours at the temperature of 550 \u003csup\u003eo\u003c/sup\u003eC. Afterward, to modify the electrode, the annealed electrode was dipped into 65% nitric acid at room temperature for 15 minutes to induce dealloying and remove the Ag material from the electrode. Finally, the modified electrode was thoroughly rinsed with DI water. To biofunctionalize the gold surface with antibody, we followed a series of steps, including, first 50 \u0026micro;L of an MAA solution (14 mmol/L) was applied to the working electrode at room temperature for 2 hours. Then, the electrode was washed with ethanol to remove any unbound MAA from the surface. To activate the carboxylic groups, a solution of 50 \u0026micro;L of 50 mM EDC/NHS (1:1) in PBS buffer at pH 6.0 put on the working electrode surface for one hour at room temperature. Next, 50 \u0026micro;L of 10 \u0026micro;g/ml VHH anti-VEGF was deposited on the activated electrode overnighted at 4 ℃ for antibody fixation. To prevent and block non-specific bindings, we applied a gelatin solution (50 \u0026micro;L of 20 mg/mL in PBS) to the electrode for 45 minutes \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Subsequently, different VEGF concentrations were introduced to the activated surface for 45 minutes at 4 ℃ to characterize the biosensor. Throughout all procedures, the electrode was kept in a dark ambient environment. After activating the carboxylic groups, any unbound reagents were washed away with a PBS solution (pH 7.4) at each stage. For a visual representation of the fabrication of the sensor, the antibody fixation and antigen detection stages are all shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Result and discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1. Characterization of the electrode surface\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates the different fabricated electrodes through alloying and dealloying processes. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea shows the morphologies of the alloying structure of Ag-Au on the FTO substrate. Due to the thermal annealing process, semi-spherical Ag-Au nanoparticles are shaped on the FTO surface, wherein a hybrid layer containing a 25 nm silver thin film and a 5 nm gold thin film were initially deposited using the PVD system. In Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, the Au nano-porous structures are visible after the dealloying process. The energy-dispersive X-ray spectroscopy (EDS) confirms the complete removal of silver during the dealloying process, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eFigures \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee show the difference in hydrophilicity of the optimized Ag-Au electrode before and after the dealloying process, as demonstrated via the contact angle measurement. The porosity of the formed gold nanostructures directly effects on the measured contact angle, as the presence of nanopores on the surface increases the hydrophilicity of the working electrode. After dealloying, the contact angle decreased from 21.1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e to 10.4\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, indicating that the surface had become more hydrophilic. This almost 50% decrease in the contact angle demonstrates improved hydrophilicity of the formed structure. Consequently, the low contact angle of the electrode surface leads to better connecting biomolecules to the electrode, enhancing the biofunctionality and detection capability of the sensor. To confirm the phases and components of the optimized electrode, XRD patterns were conducted, and the results are shown in Fig .2f. In addition, the XRD patterns of the bare FTO electrode, the bi-layer of 25 nm Ag and 5 nm Au electrode annealed at 550 \u003csup\u003eo\u003c/sup\u003eC, and the final nano-porous gold nanostructures have been shown for comparison purposes.\u003c/p\u003e\n\u003cp\u003eThe XRD patterns provide evidence for the creation Ag-Au nanoparticles, and then the porous gold particles on the FTO substrate. The peaks of Au, Ag and FTO have many similar diffraction angles leading to appearance of several broadened peaks in the XRD spectrum. The crystallographic directions of various planes have also been displayed in the spectrums. The primary crystallographic orientations for the FTO substrate correspond to the (110), (101), (200), (211), (310), and (301) planes, with corresponding 2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\theta }\\)\u003c/span\u003e\u003c/span\u003e values of 26.6\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, 33.8 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, 37.8 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, 52 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, and 62 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, respectively. We observe even stronger peaks for the gold and silver nanoparticles. The prominent peaks of Au-Ag nanoparticles are observed at 2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\theta }\\)\u003c/span\u003e\u003c/span\u003e values of 38.2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, 64.6\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, and 77.6\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e with the miller indices (hkl) of (111), (220), and (311) for the primary crystallographic orientations. After dealloying, nano-porous gold nanoparticles remain with the plane orientations of (111) and (220) at 2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\theta }\\)\u003c/span\u003e\u003c/span\u003e the angles of 37.5\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e and 43.6\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(^\\circ\\)\u003c/span\u003e\u003c/span\u003e, respectively.\u003c/p\u003e\n\u003cp\u003eThere are two important parameters in forming of porous nanostructures during the alloying and dealloying processes: the ratios of gold to silver in the PVD process and the annealing temperature in the thermal processing. Both of these parameters determine the size and porosity of the final porous nanostructures.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the FE-SEM images of fabricated gold nanostructures with different thickness ratio of Ag and Au layers after the dealloying process, at the same annealing temperature of 550 \u003csup\u003eo\u003c/sup\u003eC. The figure shows that the thickness ratio of silver to gold considerably affects the size of the dealloyed nanostructures. No observable porosity was revealed after post-dealloying in the case of 5 nm silver and 5 nm gold in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea. Conversely, as the silver thicknesses increase to 10, 15, 20, 25, and 30 nm, a corresponding boost in the porosity of the emerged post-dealloying structure, is revealed. Nonetheless, no noticeable alteration occurs for silver dimensions of 25 nm and 30 nm. This conducts us to select the case of 25 nm silver and 5 nm gold arrangement as the best initial Ag-Au thicknesses for the alloy-dealloy process.\u003c/p\u003e\n\u003cp\u003eIn continuation of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e results, the morphological structures of various arrangements involving 10 nm gold thin film integrated with different silver thicknesses of 5, 20, and 40 nm were investigated, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e (g-i). Again, no porosity was exhibited after dealloying in the case of the 10 nm gold thin film coupled with the 5 nm silver thin film. As observed previously, raised silver content correlates with increased porosity. Drawing from these figures\u0026rsquo; analysis, one can infer that the thickness of initial silver substrate compared to the finial gold over-layer plays a crucial role in initiating and boosting the porosity of the structure. Simultaneously, the amount of gold dictates the ultimate dimensions of the formed nanostructure. However, the use of more than 10 nm gold thickness was generally limited due to the increased gold consumption, which posed economic inefficiency concerns.\u003c/p\u003e\n\u003cp\u003eTo investigate and obtain an optimized annealing temperature for the thermal process, the morphologies and the average sizes of nanostructures after the dealloying process were measured as a function of the annealing temperature. As the temperature increases, the porosities become more apparent, and their surface distribution increases in the final structure. Despite this issue, according to the size distribution histograms, the overall size structure is similar for both temperatures of 550 and 600 \u003csup\u003eo\u003c/sup\u003eC. Therefore, the electrode with the annealing temperature of 550 \u003csup\u003eo\u003c/sup\u003eC with thicknesses of 25 nm silver and 5 nm gold was selected as the optimized electrode for further sensing investigations. In addition, further results of this investigation have been shown in Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e, in the supplementary information.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Electrochemical characterization of the fabricated electrodes\u003c/h2\u003e\n\u003cp\u003eIn this section, the modified electrodes were subjected to electrochemical characterization via cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The results for the bare FTO, the optimized Au-Ag electrode, and the optimized Au-Ag electrode after the dealloying process, were compared in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and b. Initially, the peak-to-peak potential of the FTO substrate was measured at 624 mV, and the maximum current was 620 \u0026micro;A. According to the EIS result, the FTO surface resistance was about 194 Ω. After modifying the FTO surface to the optimized Ag-Au layer (before the dealloying process), the silver peaks appear in the CV result at the potentials of 141 mV and 552 mV. In this case, the surface resistance reaches 56 Ω, indicating about 72% decrement relative to that of the bare FTO electrode.\u003c/p\u003e\n\u003cp\u003eIn the next step, silver was dealloyed and the porous Au nanoparticles were produced. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and b, the current peak value of the modified porous gold electrode reaches 830 \u0026micro;A, indicating about 34% increase in the current compared to the pure FTO substrate. Although the peak current of the Ag-dealloyed electrode is about 12% less than this electrode before dealloying, the peak-to-peak potential for the dealloyed electrode is about 35% less than this electrode before the dealloying process. This indicates better reversibility of the dealloyed electrode against redox reactions relative to the same case before dealloying. In addition, the dealloyed electrode has a 235 mV peak-to-peak potential which is about 33% less than that of the bare FTO. Finally, the resistance of the modified dealloyed surface relative to the FTO surface decreased by 93%, reaching about 14Ω. In addition, the stability of the electrode was investigated in the first, 20th, 50th, and 100th cycles, and it is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec that no significant change was seen after the 50th cycle. In order to have an insight into the time of the dealloying process, we measured the open circuit potential (OCP) to evaluate the impact of nitric acid dealloying on the electrode surface. As illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, after approximately 10 minutes, nearly 94% of the change of OCP has taken place. When the OCP stabilizes, it indicates that the dealloying process is fully developed and all the silver has been completely removed. In our experiments, we have chosen an alloying time of approximately 15 minutes to ensure the complete removal of silver from the electrode surface.\u003c/p\u003e\n\u003cp\u003eIn Fig. S2, the influence of the annealing temperature on the electrochemical response of the modified electrode at three annealing temperatures of 450, 550, and 600 ℃ has been investigated. The diameter of the semicircle in the Nyquist plot represents the charge transfer resistance (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{R}}_{\\text{c}\\text{h}}\\)\u003c/span\u003e\u003c/span\u003e). The \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{R}}_{\\text{c}\\text{h}}\\)\u003c/span\u003e\u003c/span\u003eat the temperatures of 450 and 600 ℃ is higher than that of 550 ℃. The same scenario is also repeated for the peak current of the 550 ℃ sample, in the CV results.\u003c/p\u003e\n\u003cp\u003eAs depicted in Fig. S3, it is apparent that the square root of the scan rate shows a clear linear relationship with the anodic peak current. This observation strongly indicates that the redox reaction involving the nano-porous gold nanostructures follow a diffusion-controlled process.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Immunosensing characterization and analytical performance\u003c/h2\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eAs previously mentioned in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the process of anchoring antibodies to the modified electrode and detecting the VEGF biomarker involves using MAA, EDC, and NHS. These compounds are utilized for immobilization on the modified electrode, facilitating the covalent attachment of antibodies to the transducer element. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the immunosensensing characterization has been performed by CV, EIS, and DPV measurements. At first, the CV, EIS, and DPV tests of the bare dealloyed electrode. we prepared these tests after immobilizing the antibody on the surface. The predominant covalent bonding of the VHH to the electrode surface leads to a further reduction in the anodic peak current to 717\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\text{A}\\)\u003c/span\u003e\u003c/span\u003e. After that, to block the empty places on the surface, we used gelatin, appearing in the increment of the electrode resistance from (56 Ω) for the antibody state to (107 Ω) after the block stage. In this case, the reduction of the current peak reads from (717 \u0026micro;A) to (610 \u0026micro;A). In the last step, VEGF antigen was placed on the surface to ensure the correct connection of all steps. As it is clear in the figure, in the last stage, which is also the antigen detection stage, the current peak in the CV diagram has decreased to (525 \u0026micro;A) in addition to about 31% increment of the peak-to-peak voltage compared to the state of Au porous. The EIS graph shows the resistance increment from (107 Ω) to (204 Ω), and the DPV diagram also shows the decrease in the current, which are all proofs of antigen detection by the desired immunosensor.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eAlso, result of the numerical values of the electrical parameters correspond to equivalent EIS circuit was used and the results of obtained parameters have been summarized in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed. Also, the equivalent circuit for modeling the EIS result has been shown in the inset of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e shows the calibration curve result of the VEGF determination by the fabricated sensor. In each step, to find the linear range, the difference between the resistances (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\varDelta {\\text{R}}_{\\text{c}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e) with the state where VEGF is not present in the solution is measured. The equation of concentrations ranges from 0.1 pg/ml to 0.1 \u0026micro;g/ml can be expressed as ∆R\u003csub\u003ect\u003c/sub\u003e(Ω)\u0026thinsp;=\u0026thinsp;84.35C\u003csub\u003eLog (VEGF)\u003c/sub\u003e [gr/ml]\u0026thinsp;+\u0026thinsp;1146.6 with a regression coefficient of 0.97. This immunosensor has a very good limit of detection of 0.1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\)\u003c/span\u003e\u003c/span\u003epg/ml. Also, in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec the selectivity of our immunosensor has been demonstrated. It shows that the interference species produce currents that are lower than 20% of the VEGF signal. This finding indicates the high-level selectivity of this sensor for detecting VEGF, as it suggests minimal interference from other substances.\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eIn Table.1, the characteristics of different biosensors for VEGF in the literature have been compared by this sensor. The findings illustrate that the nano-porous Au electrode outperforms various biosensors, especially regarding the limit of detection (LOD) and linear response range when of VEGF detection.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eTable.1: Comparison of the analytical capabilities of different immunosensors documented for the detection of VEGF with the current research.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBiosensor substrate\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eDetection method\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLinear range\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLOD\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eRef.\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\u003eAu/3-MPA/EDC-NHS/VEGF-R1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCV\u003c/p\u003e\n\u003cp\u003eEIS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u0026ndash;70\u003c/p\u003e\n\u003cp\u003epg/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e38\u003c/p\u003e\n\u003cp\u003epg/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePOLY (3,4-ethylene dioxythiophene) (PEDOT)/Gold Nanoparticle (Au NP) Composites\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEIS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u0026ndash;20\u003c/p\u003e\n\u003cp\u003epg/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.5\u003c/p\u003e\n\u003cp\u003epg/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePdPtMo CME NPs\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEIS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u0026ndash;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003epg/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.2\u003c/p\u003e\n\u003cp\u003epg/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRGO/Au NPs/11MUA/Antibody/VEGF/HRP\u003c/p\u003e\n\u003cp\u003e(Sandwich type)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCV\u003c/p\u003e\n\u003cp\u003eSWV\u003c/p\u003e\n\u003cp\u003eEIS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2-20000\u003c/p\u003e\n\u003cp\u003eng/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003cp\u003efg/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecellulose paper\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003efluorescence control (FApt)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e100\u0026ndash;5000\u003c/p\u003e\n\u003cp\u003eng/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e137\u003c/p\u003e\n\u003cp\u003eng/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAu nano porous/MAA/EDC-NHS/VHH/Gelatin/VEGF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCV\u003c/p\u003e\n\u003cp\u003eEIS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({10}^{-4}{-10}^{2}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eng/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.1\u003c/p\u003e\n\u003cp\u003epg/ml\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThis work\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, we have successfully designed an electrochemical immunosensor for detecting the VEGF tumor marker based on a modified porous gold electrode. The electrode is created through a series of straightforward processes, involving silver and gold deposition on an FTO substrate, followed by thermal annealing and dealloying to entirely eliminate silver from the electrode surface. Employing a comprehensive protocol for antibody attachment to the gold substrate, we conducted VEGF detection using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Impressively, this immunosensor exhibits a remarkable detection limit of 0.1 pg/ml, making it an ideal tool for highly sensitive VEGF tests. Furthermore, the linear range extends from 0.1 pg/ml to 0.1 \u0026micro;g/ml, indicating not only a low limit of detection but also a wide dynamic range for accurate measurements. In conclusion, this research not only establishes a robust method for sensitive electrode fabrication but also highlights its suitability for widespread adoption, positioning it as a promising candidate for scaling up in various scientific and medical contexts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.Y., H.S., R.H.S. and A.M. conceived of the presented idea and developed the theory and verified the analytical methods. S.Y., M.K. and H.S. carried out the experiments. S.Y., H.S. and A.M. wrote the manuscript. All authors discussed the results and contributed to the final manuscript. A.M. supervised the project.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work has been supported by the research council of Tarbiat Modares University (TMU). A. M. acknowledges the TMU support under grant number IG-39708.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang, S. \u003cem\u003eet al.\u003c/em\u003e Metal\u0026ndash;organic frameworks (MOFs) based electrochemical biosensors for early cancer diagnosis in vitro. \u003cem\u003eCoord. Chem. Rev.\u003c/em\u003e \u003cstrong\u003e439\u003c/strong\u003e, 213948 (2021).\u003c/li\u003e\n\u003cli\u003eSantana Oliveira, I., da Silva Junior, A. G., de Andrade, C. A. S. \u0026amp; Lima Oliveira, M. D. Biosensors for early detection of fungi spoilage and toxigenic and mycotoxins in food. \u003cem\u003eCurr. Opin. 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Acta\u003c/em\u003e \u003cstrong\u003e1199\u003c/strong\u003e, 339588 (2022).\u003c/li\u003e\n\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Electrochemical immunosensor, VEGF detection, Porous gold electrode, Fabrication methodology, Biomarker measurement ","lastPublishedDoi":"10.21203/rs.3.rs-3881185/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3881185/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe present an advanced electrochemical immunosensor designed for precise detection of the Vascular Endothelial Growth Factor (VEGF). The sensor is constructed on a modified porous gold electrode through a fabrication process involving the deposition of silver and gold on an FTO substrate. Employing thermal annealing and a de-alloying process, the silver is eliminated from the electrode, resulting in a reproducible porous gold substrate. Utilizing a well-defined protocol, we immobilize the heavy-chain (VHH) antibody against VEGF on the gold substrate, facilitating VEGF detection through various electrochemical methods. Remarkably, this immunosensor achieves an outstanding performance, featuring an impressive detection limit of 0.1pg/ml and an extensive linear range from 0.1pg/ml to 0.1\u0026micro;g/ml. This emphasizes its exceptional ability to precisely measure biomarkers across a wide concentration spectrum. The robust fabrication methodology employed in this research underscores its potential for widespread application, offering enhanced precision, reproducibility, and remarkable detection capabilities for the developed immunosensor.\u003c/p\u003e","manuscriptTitle":"Gold Nanostructure-Enhanced Immunosensing: Ultra-Sensitive Detection of VEGF Tumor Marker for Early Disease Diagnosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-29 13:52:14","doi":"10.21203/rs.3.rs-3881185/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-16T14:26:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-02-08T16:12:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41f31fd4-6499-4d3a-84ea-5c3b1e1582d4","date":"2024-01-30T01:57:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9768ee5f-1706-47d9-9460-7879f56ee793_SNPRID","date":"2024-01-30T01:56:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-30T01:52:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-25T02:24:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-01-24T18:16:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-24T17:23:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-01-20T09:33:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e8ce15b-88d5-44ae-9c5c-7b4233ea67e6","owner":[],"postedDate":"January 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28365160,"name":"Biological sciences/Biotechnology/Nanobiotechnology"},{"id":28365161,"name":"Physical sciences/Nanoscience and technology/Nanoscale devices"}],"tags":[],"updatedAt":"2024-05-09T04:00:32+00:00","versionOfRecord":{"articleIdentity":"rs-3881185","link":"https://doi.org/10.1038/s41598-024-60447-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-05-07 04:00:32","publishedOnDateReadable":"May 7th, 2024"},"versionCreatedAt":"2024-01-29 13:52:14","video":"","vorDoi":"10.1038/s41598-024-60447-2","vorDoiUrl":"https://doi.org/10.1038/s41598-024-60447-2","workflowStages":[]},"version":"v1","identity":"rs-3881185","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3881185","identity":"rs-3881185","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}