Fabrication of electrochemical aptasensor based on gold nanoparticles and P53 graphene oxide for protein detection

Abstract

Recently, aptamers have been considered in cancer diagnosis and treatment applications by binding to various bioreceptor molecules. Given that the p53 protein is responsible for approximately 50% of cancers, its identification is promising, but so far, no aptamer DNA sequence has been designed for the p53 protein and aptamer-based sensor. The purpose of this invention is to develop a nanoparticle-based aptamer sensor and use the p53 bioreceptor for cancer diagnosis with high sensitivity in the shortest time. In this regard, a DNA aptamer-based sensor was used on a glassy carbon electrode coated with gold nanoparticles / graphene oxide / conductive polymer . This sensor was created by the gold nanoparticles / aptamer-toluidine blue enhancer. The results obtained under optimal conditions of electrochemical investigation using amperometry indicated a detection limit of 0.29 picograms / mL in phosphate buffer and 0.341 picograms / mL in human blood plasma and a linear range of 1 to 300 picograms / mL for the sensor. This invention demonstrates the excellent and controllable performance of a modified carbon electrode to create a suitable sensor for detecting the cancer marker p53 protein.

Description

Description of the invention Title of the invention (as stated in the declaration) Fabrication of electrochemical aptasensor based on gold nanoparticles and graphene oxide for detection of p53 protein Technical background of the relevant invention Rapid diagnostic tools: nanobiosensors. Technical problem and stating the objectives of the invention Given the critical importance of the p53 protein, which is observed in more than 50% of cancer types, the development of accurate, rapid, and reliable tools for detecting this protein is of particular importance. Despite significant advances in the field of electrochemical sensors, there are still several challenges, including insufficient sensitivity in detecting very low amounts of the target analyte; limited selectivity in the presence of interfering molecules; long response times, and complex and expensive fabrication processes that require specialized equipment. These problems have hindered the widespread and cost-effective application of electrochemical sensors in medical diagnostics. In addition, the DNA (Deoxyribonucleic acid) aptamer sequence used in this invention has been designed and used for the first time, which has significantly improved the sensitivity and efficiency of the sensor. In order to address these challenges, the research objectives were defined as follows: Development of a low-cost electrochemical biosensor for accurate detection of p53 protein; Surface modification of glassy carbon electrode using gold nanoparticles and graphene oxide; Design of a sandwich model of two specific aptamers for target recognition; Using gold nanoparticles functionalized with toluene blue as an electrooxidation agent for signal amplification; Optimizing biosensor manufacturing steps to improve performance; Detection of p53 protein in real human serum samples using amperometric technique A description of the state of the prior art and the history of developments related to the claimed invention. Library studies show that although numerous studies have been conducted on the development of electrochemical sensors for the detection of p53 protein, there are still limitations in sensitivity and ease of fabrication. For example, Kang et al. (2021) used gold nanoparticles and conductive polymer as active substrates and zeolite imidazolate frameworks as signal amplifiers in an antigen-antibody sandwich sensor and reported a detection limit of 0.09 ng / mL. In another report, titled Electrochemical detection of different p53 conformations by using nanostructured surfaces, by Tonello and colleagues in 2019, they used nanostructures such as carbon nanotubes and platinum nanoparticles, and reported the best detection limit of 1 nanogram / mL in the presence of platinum nanostructures. Also in 2013, Shwetha et al., in an article titled Aptamer–nanoparticle-based chemiluminescence for p53 protein, developed an RNA (Ribonucleic acid) aptasensor using gold nanoparticles-based chemiluminescence assay, which reported a protein detection limit of 0.1 ng / ml (color change from yellow to purple). The catalytic activity due to agglomeration of gold nanoparticles was enhanced by chemiluminescence, and the detection limit was achieved at 10 pg / ml. A Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention As mentioned, various methods have been used to detect p53 protein. The use of nanomaterials has been used as an upgrade of sensors due to their high surface area to volume ratio and enhanced signal response. However, due to the stability and high sensitivity of aptamer-based sensors to antigen and the lack of DNA aptamer sequence, an aptasensor for this protein was not developed. DNA aptamers are more stable than RNA aptamers due to their non-degradability. In this study, DNA aptamers are immobilized on a glassy carbon electrode modified with gold nanoparticles and graphene oxide to detect p53 protein, and gold nanoparticles modified with ToluidineBlue (TBO) and aptamers have been used as signal enhancers. Steps and method of execution: 1) Construction of electrochemical optosensor: In order to make electrochemical aptasensor, glassy carbon electrode was used as working electrode and reference electrode was made of silver / silver chloride saturated with potassium chloride and platinum counter electrode was used. Gold nanoparticles were prepared by overpotential method at -0.6 V potential and after immobilization of graphene oxide and conductive polymer, amine modified aptamer is immobilized on the surface. In this system, the sample volume used in 8 microliters of protein is placed on the surface and finally gold nanoparticles-aptamer-TBO solution which is formed by amine aptamer and TBO on the modified surface agent of gold nanoparticles is dripped on the electrode and cyclic voltammetry test is drawn and read in the range of -0.6 to 0.6 V in 0.1 M ferro / ferricyanide and potassium chloride electrolyte solution. The electrochemical device used is Autolab device, which has been used as a precise device for electrochemical method. 2) Synthesis of nanoparticles: Gold nanoparticles are synthesized by the overpotential method at a potential of -0.6 V in a chloroaric acid solution on the surface of a glassy carbon electrode. 3. Stabilization of graphene oxide and conductive polymer on the surface of gold nanoparticles After washing the electrode with acetonitrile solution, 1 M graphene oxide was placed on the electrode surface and dried at ambient temperature. Then, the prepared electrode was placed in a 1 mM solution of thiophene monomer (TBA) with acetonitrile solvent and tetrabutylammonium perchlorate (0.1 M) to perform polymerization by two cyclic voltammetry in the range of 0 to 1.4 V at a rate of 100 mV / s using a silver / silver chloride reference electrode. 4. Fixation of the aptamer on the modified electrode After washing the electrode with acetonitrile to remove free monomers, the prepared electrode was placed in a mixture of N-hydroxysuccinamide ester (NHS) / N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDC) (10 mM / 20 mM) for 6 hours in a shaking incubator at 37°C to activate the carboxyl functional groups on the surface. Then, the optimal concentration of the capping aptamer with the sequence 3-TCCGAGTCTAGAAGCGGACATGCCCGGGCATGTCCTGTTGTAAGC-5 (2 μM) was dropped onto the electrode and kept at 4°C for 6 hours to stabilize the benzoic acid functional groups (Figure 1). 5. Protein stabilization The modified electrode was washed with phosphate buffer solution and then exposed to different concentrations of protein for the optimal time (10 minutes) to stabilize it on the electrode. 6. Making the booster solution In order to enhance the output response, an enhancer nanostructure was used. Initially, gold nanoparticles, 10 mM chloroauric acid were mixed with 10 mM trisodium citrate for one minute, and the reducing agent sodium borohydride (0.1 M) was added dropwise under gentle stirring over a period of 5 minutes. A colorless solution of TBA monomer with acetonitrile solvent (1 mM) is slowly added to the gold nanoparticle solution at a volume ratio of ½ and a change in color from red to purple is observed (Figure 2). After self-assembly of gold / TBA nanoparticles, the mixture of EDC and NHS solution in phosphate buffer was prepared and a colorless solution was obtained. The prepared solution was centrifuged twice for 5 minutes at 15,000 rpm to reduce the size of the particle clusters. Then, gold / TBA nanoparticles were added to EDC / NHS and kept in an incubator. After this time, both solutions of toluidine blue (1 mg / ml) with acetonitrile solvent and the amplified aptamer with the sequence 3-TCCGATGGCCCTGCAGTCTG were added. AGGACAATTCCTGCGCCATTCTAC-5 (2 μM) was added to the final solution and kept at 4°C for 15 min. The transmission electron microscope image and dynamic light diffraction and X-ray diffraction map are shown in Figure 3-5. 7. Fixing the amplifier on the modified electrode After protein immobilization on the modified electrode surface, the gold nanoparticle / TBA / toluidine blue-aptamer enhancer is deposited on the surface (Figure 6). The field emission scanning electron microscope image at each step and the X-ray photoelectron spectrum are shown in Figures 7 and 8. At this stage, the electrochemical aptasensor is completed in terms of surface preparation steps. 8. Detection of p53 protein 1.8. Optimization of the applied potential on the surface of the modified glassy carbon electrode: Initially, the effect of applied potential on the steady-state current was investigated by amperometry in the range of 0 to 50 seconds using a silver / silver chloride reference electrode in a nitrogen-saturated phosphate buffer solution. The voltage versus current intensity curve of the electrode showed that the voltage of -350 mV had the highest current reduction (Figure 9). 2.8. Evaluation of electrochemical optosensor: After each step of electrode surface modification, the electrochemical changes in the electrode surface were investigated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods (Figures 11 and 10). 3.8. Investigation of electrochemical changes of the electrochemical aptasensor in the presence of different concentrations of p53 protein: The amperometric test is performed in phosphate buffer medium in the presence of the electrode with different concentrations of 0, 1, 25, 50, 100, 200 pg / ml of protein serially diluted in phosphate buffer (Figure 12). 4.8. Determination of the detection limit of the biosensor in a real sample (human serum): A broad linear scaling and sensitivity plot of the electrochemical optosensor performance was performed under optimal conditions (Figure 13). 5.8. Investigation of the selectivity of the electrochemical aptasensor in the presence of interfering molecules: To investigate the selectivity of this aptasensor, an amperometric method was used to detect p53 in phosphate buffer. Interfering molecules including cystine, tyrosine, ascorbic acid, uric acid and dopamine were used at twice the concentration of p53 protein (Figure 14). As can be seen, the intensity of the response of p53 protein and its mixture is higher than that of interfering molecules, indicating good stabilization of p53 protein and high selectivity of the sensor under the influence of DNA aptamers compared to interfering molecules on the electrode surface. 6.8. Checking the stability of the optosensor: In order to demonstrate the stability of the electrode, the optosensor was stored at 4°C and amperometric tests were performed every two days. After twenty days, the results of the current response were plotted against time. The results showed that after 7 days, the sensor response had decreased by 92% and after 20 days, by 78% compared to the first day. (Figure 15) Explanation of shapes, maps and diagrams Figure 1. Schematic of the manufacturing process of the modified gold nanoparticle electrode sensor without applying a reinforcing element. Figure 2. A) Gold nanoparticle solution, B) Gold nanoparticle-monomer self-assembled solution. Figure 3. Energy-filtered transmission electron microscopy images of a) gold nanoparticles, b) self-assembly of monomer and gold nanoparticles, and c) reinforcing element. Figure 4. a) Zeta potential analysis of gold nanoparticles, measurement of average particle size for b) gold nanoparticles, c) monomer / gold nanoparticle self-assembly and d) reinforcing element by dynamic light diffraction. Figure 5. X-ray energy diffraction map of the gold element map (a) in the gold nanoparticle solution at the scale of 90 nm and the gold element map (b) and carbon (c) and oxygen (d) in the self-assembled gold nanoparticle / TBA solution at the scale of one micrometer. Figure 6. Schematic of the steps for manufacturing a reinforcing element. Figure 7. Field emission electron microscopy images of the surface of a) gold nanoparticles, b) gold nanoparticles / graphene oxide, c) gold nanoparticles / graphene oxide / TBA, d) gold nanoparticles / graphene oxide / TBA / immobilized aptamer / protein, and e) gold nanoparticles / graphene oxide / TBA / immobilized aptamer / protein / enhancer. Figure 8. X-ray photoelectron spectra for the peaks of a) gold, b) C1s, c) N1s, d) S2p, and f) P2s for the layer (i) gold, (ii) gold nanoparticles / graphene oxide, (iii) gold nanoparticles / graphene oxide / TBA, (iv) gold nanoparticles / graphene oxide / TBA / stabilized aptamer, and (v) gold nanoparticles / graphene oxide / TBA / stabilized aptamer / protein / enhancer. Figure 9. a) Amperometric curve of the modified electrode surface at different potentials, b) Current calibration curve versus potential with nitrogen gas application. Figure 10. Cyclic voltammetry diagram of each step of the modified electrode in ferrocyanide solution (with a scan rate of 100 mV / s). Figure 11. Nyquist curve of the modified glassy carbon electrode in 5 mM ferrocyanide solution, inset: equivalent circuit of the electrochemical cell. Figure 12. A) Amperometric curve and B) protein calibration at different protein concentrations in phosphate buffer solution. Figure 13. A) Amperometric curve and b) protein calibration at different protein concentrations in human blood plasma. Figure 14. a) Amperometric curve of the modified electrode surface, b) magnitude of the decreasing current to evaluate the selectivity of the proposed aptasensor in the presence of protein, cystine, tyrosine, ascorbic acid, uric acid and dopamine. Figure 15. (a) Amperometric curve and (b) time-dependent current decay curve for evaluating the stability of the aptasensor stored at 4°C. A clear and precise statement of the advantages of the claimed invention over prior inventions. A review of previous patents shows that no patent has been registered so far in the field of designing and manufacturing an electrochemical aptasensor based on gold nanoparticles and graphene oxide, using the sandwich model for the detection of 53p protein. Among the prominent advantages of the introduced aptasensor over existing laboratory methods, the following can be mentioned: High sensitivity: By using the sandwich model and using specific aptamers along with signal enhancing agents, the detection limit has been reduced to 0.341 pg / mL in human serum samples. Small sample volume: Only 8 microliters of human serum sample is sufficient for analysis, allowing for rapid and minimally invasive diagnosis. Low cost: The use of DNA aptamers instead of common antibodies and the simple design of the sensor have significantly reduced the cost of its manufacture and development compared to classical methods. High selectivity: The use of specific aptamers and nanostructures has led to increased selectivity against other interfering molecules. Accurate evaluation of fabrication steps: Advanced electrochemical techniques including electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used in all stages of electrode surface modification, ensuring high process accuracy. Overall, this aptasensor can be used as a new route in the development of sensitive and reliable biosensors for early detection of cancers. Description of at least one implementation method for implementing the invention The presented electrochemical aptasensor is capable of detecting and quantifying p53 protein in real human plasma samples. After sample preparation, the aptasensor is incubated in the sample under optimal conditions for about 8 minutes. Then, the presence of p53 protein is analyzed using a potentiostat / galvanostat device by monitoring the changes in amperometric signals. To evaluate the sensor performance in real samples, 8 μL of human serum is dropped onto the aptasensor surface and amperometric measurements are performed. The electrochemical response is recorded and analyzed, and the concentration of p53 protein is determined within physiological and pathological ranges. Explicit mention of the industrial application of the invention The present invention is an electrochemical optosensor based on gold nanoparticles and conductive polymer, designed to identify and measure the p53 protein. This sensor, with a similar function to glucometer devices and using the amperometric method, has the ability to detect quickly, accurately, and be portable. The features of this system include high sensitivity, appropriate selectivity against interfering molecules, and the need for a very small sample volume (less than 10 microliters). Thus, this optosensor can be used as an effective tool for the early diagnosis of cancers and monitoring the progression of the disease in patients. An increase in the amount of p53 protein in biological samples indicates the development of the cancer process, and its early detection can play a vital role in preventing tumor growth and initiating effective treatment. Therefore, the present invention has direct and effective industrial application in the medical industry, laboratory diagnostics, and health monitoring, especially in the diagnosis of a wide range of cancers. Brief description of the invention

Claims

Claim What is claimed: What is claimed is an electrochemical aptasensor system comprising: a nucleic acid-specific aptamer immobilized on a conductive substrate modified with a metal nanoparticle and a two-dimensional carbon material, a signal amplification system comprising a metal nanoparticle attached to the aptamer and an interacting molecule having active functional groups, which is used for electrochemical detection and measurement of a protein biomarker. Claim 2 According to claim 1 The system according to any one of the preceding claims, wherein the two-dimensional carbon material comprises graphene oxide and the metal nanoparticle comprises gold nanoparticles. Claim 3 In accordance with claim 1, the system according to any one of the preceding claims, the electrode substrate of which comprises a glassy carbon electrode, an auxiliary electrode made of platinum and a reference electrode made of Ag / AgCl saturated with a potassium chloride solution. Claim 4 is in accordance with claim 1 of the system according to any one of the preceding claims, wherein the interacting molecule comprising toluidine blue as the redox agent has a free amine group capable of reacting with the carboxyl groups of the surface modified with gold nanoparticles.

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