Nanocomposite material, glassy carbon modified electrode, electrochemical sensor and preparation method

Abstract

The invention relates to a nano composite material, a glassy carbon modified electrode, an electrochemical sensor and a preparation method, and relates to the technical field of electrochemical sensing detection. Comprising the following steps: oxidizing carbon nano onions, and sequentially mixing the obtained oxidized carbon nano onions with methylene blue and gold nanoparticles, so as to obtain an AuNPs-MB-oxCNOs nano composite material; the preparation method comprises the following steps: modifying the surface of a glassy carbon electrode with an AuNPs-MB-oxCNOs nano composite material to obtain a carbon nano composite layer; and modifying the milt DNA on the carbon nano composite layer to obtain the glassy carbon modified electrode. According to the invention, methylene blue and gold nanoparticles are connected to oxidized carbon nano onion in a non-covalent manner, the prepared AuNPs-MB-oxCNOs nano composite material is used as a biocompatible matrix of milt DNA, and the PRO sensing electrochemical sensor is constructed by detecting a reduction value of an MB current signal based on adsorption of PRO on the milt DNA.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensing and detection, and particularly to nanocomposites, glassy carbon modified electrodes, electrochemical sensors and preparation methods thereof. Background Art

[0002] Propranolol (PRO, 1-(isopropylamino)-3-(1-naphthyloxy)-2-propanol) belongs to the class of β-blockers and is widely used in the treatment of hypertension, angina pectoris and myocardial infarction. However, the effects of PRO in reducing heart rate, reducing anxiety, improving concentration and performance have led to its abuse in competitions that require concentration. The World Anti-Doping Agency has clearly stated that PRO is a substance prohibited for athletes. Therefore, the selective and sensitive detection of PRO is crucial for ensuring the fairness of competitive sports. In the quantitative detection of PRO, a large number of analytical methods have been developed and reported, including optical methods, capillary electrophoresis and various chromatographic techniques, etc. Although these analytical methods have shown excellent sensitivity, the expensive test cost and complex pretreatment steps limit the application of these analytical methods.

[0003] Compared with the above methods, electrochemical technology has the advantages of fast response, high sensitivity, low cost, etc., which makes it a choice for determining PRO without sample separation procedures. The structural feature of PRO is that naphthalene diphenoxy is connected to an alkyl chain with amino and hydroxyl functional groups. Although PRO is electroactive and can undergo electrochemical transformation, the electron transfer rate of this step is slow, so it is difficult to directly obtain the electrochemical response signal of PRO. Therefore, the development of novel nanocomposites for constructing high-performance PRO electrochemical sensors remains a great challenge.

[0004] Methylene blue (MB) is a derivative of phenothiazine dyes and can be used as an electrochemically redox-active indicator to detect the reaction process in biosensors and indirectly detect target chemical molecules. The excellent electrocatalytic performance of MB can effectively improve the electron transfer between the analyte and the electrode. In view of this, the present invention provides nanocomposites, glassy carbon modified electrodes, electrochemical sensors and preparation methods thereof. Summary of the Invention

[0005] The technical problems to be solved by the present invention are to provide nanocomposites, glassy carbon modified electrodes, electrochemical sensors and preparation methods thereof. The aim is to construct an electrochemical sensor fDNA / AuNPs-MB-oxCNOs / GCE for sensing propranolol (PRO), provide a novel redox platform with high electrochemical activity and conductivity, and realize the sensitive, accurate and convenient detection of PRO.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] First aspect, a method for preparing a nanocomposite, comprising the following steps:

[0008] Oxidize carbon nano-onions (CNOs) to obtain oxidized carbon nano-onions (oxCNOs); perform a first mixing of the oxidized carbon nano-onions with methylene blue (MB) to obtain an MB-oxCNOs nanomaterial; perform a second mixing of the MB-oxCNOs nanocomposite with gold nanoparticles (AuNPs) to obtain an AuNPs-MB-oxCNOs nanocomposite.

[0009] Among them, MB has a planar rigid aromatic ring structure and is positively charged, and can be tightly adsorbed onto the oxCNOs structure through electrostatic and π-π stacking interactions to form a nanocomposite. CNOs is a unique carbon allotrope composed of onion-like multi-layer concentric graphite shells, having a large specific surface area, good electrical conductivity, chemical stability, and excellent biocompatibility, showing great application potential in constructing electrochemical biosensors; introducing oxygen-containing functional groups (such as carboxylic acid functional groups) into CNOs can further regulate its electrostatic properties, conjugated network, and surface defects; the special spatial structure and oxygen-containing functional groups on the surface of oxCNOs can provide multiple active sites for chemical reactions. oxCNOs is not only composed of sp 2 carbons with high π electron density, but also has high hydrophilicity, is negatively charged under neutral conditions, and the positively charged MB molecules can be attached to the negatively charged oxCNOs through electrostatic interactions. The polynuclear aromatic complex MB can be assembled on the graphite shell of oxCNOs to form an MB-oxCNOs nanocomposite, combining the advantages of MB and CNOs; AuNPs have high catalytic performance due to their small particle size and high chemical stability, and the excellent properties of oxCNOs make it suitable for loading AuNPs, which can prevent the aggregation of AuNPs and endow the metal nanostructured carbon material composite with better catalytic activity.

[0010] The beneficial effects of the present invention are: MB and AuNPs of the present invention are non-covalently linked to oxCNOs to prepare an AuNPs-MB-oxCNOs nanocomposite. Detection and analysis show that MB and AuNPs are uniformly dispersed in the oxCNOs matrix; when manufacturing a novel PRO electrochemical sensor with a signal amplification function, the AuNPs-MB-oxCNOs nanocomposite is used as a biocompatible matrix for fDNA. Based on the adsorption of PRO on fish sperm DNA (fDNA), by detecting the decrease value of the MB current signal, a PRO electrochemical sensor (fDNA / AuNPs-MB-oxCNOs / GCE) is constructed, providing a novel redox platform with high electrochemical activity and conductivity.

[0011] On the basis of the above technical solutions, the present invention can also be improved as follows.

[0012] Further, the oxidizing agent used for oxidation includes at least one of nitric acid and sulfuric acid; the sulfuric acid is concentrated sulfuric acid with a mass fraction of ≥95%; the nitric acid is concentrated nitric acid with a mass fraction of ≥65%;

[0013] The mass ratio of the carbon nano-onions to the oxidizing agent is 30 - 36:14, preferably 33:14;

[0014] The mass ratio of the oxidized carbon nano-onions to the methylene blue is 1:1 - 2, preferably 1:1.5;

[0015] The mass ratio of the MB-oxCNOs nanocomposite to the gold nanoparticles is 1:0.1 - 2, preferably 1:1.

[0016] Further, the parameters for oxidation are: the temperature is 100°C - 110°C, and the time is 5h - 15h;

[0017] The parameters for the first mixing are: the ultrasonic frequency is 40KHz - 80KHz, preferably 68KHz, and the time is 1h - 3h;

[0018] The parameters for the second mixing are: the stirring rate is 200rpm - 600rpm, preferably 400rpm, and the time is 12h - 24h.

[0019] In the second aspect, a nanocomposite, the nanocomposite is prepared by the preparation method described above.

[0020] Further, the average particle size of the oxidized carbon nano-onions in the nanocomposite is 4.84nm; the average particle size of the gold nanoparticles in the nanocomposite is 14.5nm.

[0021] In the third aspect, a glassy carbon modified electrode, the glassy carbon modified electrode (GCE) includes a glassy carbon electrode and a carbon nanocomposite layer and a protamine DNA (fDNA) layer sequentially loaded on the glassy carbon electrode, and the material of the carbon nanocomposite layer includes the nanocomposite described above.

[0022] The beneficial effects of the above solution of the present invention are as follows: The present invention has successfully constructed a chemically modified glassy carbon electrode based on protamine DNA and a nanocomposite material loaded with AuNPs-MB-oxCNOs, namely a glassy carbon modified electrode, for detecting PRO; due to the excellent electrochemical properties of the fDNA / AuNPs-MB-oxCNOs nanocomposite material, such as distinct redox peaks and a high electroactive surface area, a simple electrochemical sensor has been constructed using the fDNA / AuNPs-MB-oxCNOs composite material, and PRO is detected by differential pulse voltammetry. The characterization results of SEM show the surface morphology of the modified fDNA / AuNPs-MB-oxCNOs / GCE.

[0023] Fourthly, a preparation method of the glassy carbon modified electrode includes the following steps: first, the above-mentioned nanocomposite material is modified on the surface of the glassy carbon electrode to form a carbon nanocomposite layer; then, protamine DNA is modified on the carbon nanocomposite layer to form a protamine DNA layer, and the glassy carbon modified electrode is obtained.

[0024] The beneficial effects of the above solution of the present invention are as follows: The preparation method is simple, feasible, and convenient for large-scale production.

[0025] Furthermore, the mass ratio of the nanocomposite material to the protamine DNA is 5:2 - 6, preferably 5:4.

[0026] Fifthly, an electrochemical sensor, and the electrochemical sensor includes the above-mentioned glassy carbon modified electrode.

[0027] The beneficial effects of the above solution of the present invention are as follows: The present invention has prepared a simple PRO electrochemical sensor based on protamine DNA and a carbon nanosized onion loaded with gold nanoparticles and methylene blue. The linear response range of this sensing platform (fDNA / AuNPs-MB-oxCNOs / GCE) to PRO is 0.1 nM - 100 μM, and the detection limit is 33 pM. The sensor of the present invention has good selectivity, stability, and repeatability, and has been successfully used to detect PRO in biological fluid samples, showing a broad prospect for electrochemical sensing devices.

[0028] Furthermore, the electrochemical sensor further includes a reference electrode and a counter electrode. The reference electrode is, for example, a saturated calomel electrode (SCE), and the counter electrode is a Pt electrode. Description of the Drawings

[0029] Figure 1 It is a manufacturing and sensing process diagram of the fDNA / AuNPs-MB-CNOs / GCE of the present invention;

[0030] Figure 2Characterization diagrams of the nanocomposites of the present invention; among them, (a) TEM diagram of AuNPs-MB-oxCNOs; (b) HRTEM diagram of AuNPs-MB-oxCNOs; (c) SEM-EDX of AuNPs-MB-oxCNOs;

[0031] Figure 3 Statistical diagrams of the particle sizes of oxCNOs and AuNPs in the AuNPs-MB-oxCNOs prepared according to the present invention; among them, (a) is AuNPs, and (b) is oxCNOs;

[0032] Figure 4 Detectability diagram of the fDNA / AuNPs-MB-oxCNOs / GCE electrochemical sensor of the present invention; among them, (a) shows the change of the peak current of DPV with the change of PRO concentration, and a to g are 0.10 nM, 1.00 nM, 10.0 nM, 100 nM, 1.00 μM, 10.0 μM, and 100 μM respectively; (b) is the linear diagram of the change of current with the logarithm of PRO concentration;

[0033] Figure 5 Result diagram of the anti-interference ability of the present invention. Detailed implementation manners

[0034] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased through regular channels.

[0035] Examples

[0036] 1. Description of experimental materials and instruments.

[0037] (1) Materials and reagents:

[0038] Fish sperm DNA (fDNA), brand Macklin, product number D8822137-1g, purchased from Beijing Yancheng Technology Co., Ltd.; methylene blue (MB), chloroauric acid tetrahydrate (HAuC l4 ·4H2O, 99.99%), propranolol (PRO), HNO3, H3PO4, H3BO3, Na2HPO4, NaH2PO4, NaOH, HCl, KCl, CaCl2, MgCl2, LiClO4, Na2SO4, K3[Fe(CN)6], ascorbic acid (AA), acetic acid (HAc), L-cysteine (L-cysteine), and glucose (Glu) were purchased from Xilong Chemical Co., Ltd.

[0039] The preparation method of carbon nano-onions (CNOs) can be found in the reference: Bian, Y.; Liu, L.; Liu, D.; Zhu, Z.; Shao, Y.; Li, M. Electrochemical synthesis of carbon nanoonions. Inorg. Chem. Front. 2020, 7, 4404 - 4411.

[0040] Brittone - Robinson buffer (BRB) solution was used as the supporting electrolyte. A 0.04 M BRB solution was obtained by mixing 0.04 M HAc, 0.04 M phosphoric acid, and 0.04 M boric acid, and then it was adjusted to the desired pH value by adding 0.2 M NaOH solution as the supporting electrolyte. A 1.0 mM PRO stock solution was prepared using 0.04 M BRB (pH 7.0) and stored refrigerated in a dark glass bottle. Optical measurement results showed that the purity of fDNA was high enough (OD260 / OD280 higher than 1.8, where OD represents optical density) for direct use. Double - distilled water was used throughout the experiment.

[0041] All reagents used in this invention are of analytical grade and do not require further purification.

[0042] (2) Instruments:

[0043] All electrochemical measurements, such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV), were carried out on a CHI 660D electrochemical workstation (Chenhua Co., Ltd., Shanghai, China) at room temperature.

[0044] Transmission electron microscopy (TEM) was studied using a JEOL - JEM - 2100F (JEOL, Japan) at an acceleration voltage of 200 kV.

[0045] Scanning electron microscopy (SEM) images and energy - dispersive X - ray spectroscopy (EDX) were collected on an S - 4800 electron microscope (Hitachi, Japan).

[0046] The pH value was measured using a pH meter (MP 230, Mettler - Toledo, Switzerland).

[0047] UV - vis absorption spectra were measured using a UV - vis spectrophotometer (Shimadzu UV - 3600).

[0048] 2. Experimental methods.

[0049] 2.1 Preparation of fDNA / AuNPs - MB - oxCNOs modified glassy carbon electrode (GCE) sensor.

[0050] The fabrication and sensing process of fDNA / AuNPs-MB-CNOs / GCE are as Figure 1 shown as follows:

[0051] (1) The original CNOs were ultrasonically dispersed in 10 mL of concentrated nitric acid solution with a concentration of 3.3 mg / ml for 10 minutes, and oxidized at 110 °C for 10 hours. After cooling to room temperature, they were washed three times with deionized water to remove residual reagents until the pH value became neutral, and then dried under vacuum to obtain oxidized carbon nano-onions (oxCNOs).

[0052] (2) oxCNOs (1.0 mg) and MB (1.5 mg) were dissolved in 10 mL of distilled water and ultrasonically treated for 2 hours, and then the excess MB was removed with distilled water to prepare a 0.5 mg / ml MB-oxCNOs solution, thus synthesizing the MB-oxCNOs nanocomposite.

[0053] (3) 2 ml of the prepared 0.5 mg / ml MB-oxCNOs nanocomposite was mixed with 2 ml of 0.01% AuNPs solution under vigorous stirring overnight to prepare the AuNPs-MB-oxCNOs nanocomposite. And the prepared nanocomposite was characterized.

[0054] (4) Before modification, the bare glassy carbon electrode (GCE) was polished successively with 0.3 μM and 0.05 μM alumina slurries, and then ultrasonically treated three times in deionized water to remove any residual polishing reagents. 8.0 μL of 0.5 mg / ml AuNPs-MB-oxCNOs was dropped onto the bare GCE and dried at room temperature. Then, 8.0 μL of 0.4 mg / ml fDNA solution was dropped to obtain the fDNA / AuNPs-MB-oxCNOs modified GCE sensor.

[0055] 2.2 Study on sensor characteristics.

[0056] The electrochemical behavior of the fDNA / AuNPs-MB-oxCNOs / GCE modified glassy carbon electrode sensor was studied using different electrochemical techniques, including CV, chronoamperometry and DPV, in 0.04 M BRB (pH 7.0) or 0.1 M KCl containing 1.0 mM K3[Fe(CN)6]. Among them, fDNA / AuNPs-MB-oxCNOs / GCE, Pt electrode and saturated calomel electrode (SCE) were used as the working electrode, counter electrode and reference electrode respectively. The glassy carbon modified electrode was immersed in the PRO solution and stirred for 30 minutes, and then rinsed with distilled water. Among them, the DPV method was used, with an amplitude of 50 mV, a pulse width of 60 ms, a sampling width of 20 ms, and a pulse period of 0.5 s.

[0057] 2.3 Detection of PRO in actual samples.

[0058] (1) Urine samples: Synthetic urine samples (Reference: Laube, N.; Mohr, B.; Hesse, A. Laser-probe-based investigation of the evolution of particle size distributions of calcium oxalate particles formed in artificial urine. J. Cryst. Growth 2001, 233, 367 - 374.). The samples were evaluated and analyzed by recovery rate.

[0059] (2) Human plasma samples: Human blood samples were centrifuged at 2000 rpm for 10 minutes, and the supernatant was diluted 10 times with 0.04 M BRB buffer (pH = 7.0). The plasma samples were evaluated and analyzed by recovery rate.

[0060] 3. Results and discussion.

[0061] 3.1 Characterization of nanomaterials.

[0062] The TEM images of the prepared AuNPs-MB-oxCNOs showed that the average particle size of oxCNOs particles was 4.84 nm, and the average particle size of AuNPs particles was 14.5 nm (a and Figure 2 in Figure 3 a and b). The HRTEM images showed that the oxCNOs nanoparticles were composed of concentric graphite shells with an interlayer spacing of 0.346 nm, which could be attributed to the (002) crystal plane of graphite. The lattice fringes of AuNPs corresponded to the (111) atomic plane with an interplanar spacing of 0.208 nm ( Figure 2 b). In the elemental mapping images of AuNPs-MB-oxCNOs, the elements C, O, S, N, and Cl were evenly dispersed, which indicated that CNOs had been successfully oxidized to oxCNOs, and MB was evenly adsorbed on the surface of oxCNOs. The gold elemental map was consistent with the STEM results, indicating that gold nanoparticles were evenly dispersed in the homogeneous matrix oxCNOs in the form of single particles. These results verified the successful synthesis of AuNPs-MB-oxCNOs ( Figure 2 c).

[0063] This study investigated the parameters influencing MB adsorption on oxCNOs. The surface of oxCNOs carries a significant negative charge, while MB is positively charged, making it highly susceptible to MB adsorption via electrostatic interactions. The negatively charged oxCNOs are well-suited for loading negatively charged gold nanoparticles, which prevents aggregation and imparts enhanced catalytic activity to nanometal-reinforced carbon-based composites.

[0064] 3.2 Analytical performance of fDNA / AuNPs-MB-oxCNOs / GCE for PRO.

[0065] Electrodes and differential pulse voltammetry (DPV) techniques were applied to PRO sensing. Figure 4 As shown in Figures a and b, the electrochemical response of PRO was tested on fDNA / AuNPs-MB-oxCNOs / GCE. After incubation of fDNA / AuNPs-MB-oxCNOs / GCE in different concentrations of PRO for 30 minutes, the peak current gradually decreased, indicating that the electrochemical response of the electrode was affected by PRO.

[0066] Hypothesizing that the interaction between fDNA and PRO might generate an fDNA-PRO complex, the anionic sugar-phosphate backbone of DNA can bind to the small molecule PRO, which has a planar, rigid aromatic ring structure, through electrostatic, hydrogen-bonding, and hydrophobic interactions to form a complex. Because PRO strongly interacts with DNA, it creates a barrier to electron transfer at the electrode surface. When the DNA-modified electrode was immersed in a solution containing PRO, the interaction of PRO with the fDNA / AuNPs-MB-oxCNOs / GCE sensing platform showed a decrease in the current response.

[0067] In the range of 0.10 nM to 100 μM, the change in current versus the logarithm of PRO concentration was linear, with the regression equation being I / μA = -2.965 lg (CPRO / M) + 5.454 (R2 = 0.9913). The detection limit of PRO was 33 pM.

[0068] Compared with existing electrochemical sensors (Table 1), fDNA / AuNPs-MB-oxCNOs / GCE has a wider linear range (0.10 nM to 100 μM) and a lower detection limit (33 pM). The high sensitivity of fDNA / AuNPs-MB-oxCNOs / GCE for PRO detection may be attributed to the following aspects: (1) Under neutral conditions, negatively charged oxCNOs are loaded with positively charged MB through electrostatic interaction; the curved graphene plane may interact with MB through π-π, which may contribute to the adsorption of MB on oxCNOs. (2) The efficient electrocatalytic properties of AuNPs, as well as the uniform distribution of AuNPs catalysts on oxCNOs and within the MB-oxCNOs substrate. (3) MB adsorbed on oxCNOs can interact with fDNA, enhancing the stability of the sensing interface. The large specific surface area of fDNA / AuNPs-MB-oxCNOs can amplify the response current. (4) The interaction between fDNA and PRO creates a barrier for electron transfer on the electrode surface, thus generating the signal of the PRO sensor. Therefore, the fDNA / AuNPs-MB-CNOs / GCE proposed in the present invention can be used for the determination of trace PRO in drug samples.

[0069] Table 1 Performance of reported electrochemical sensors for the detection of PRO

[0070]

[0071] References [1]-

[11] in Table 1 are detailed in the following records:

[0072] [1] Kun, Z.; Hongtao, C.; Yue, Y.; Zhihong, B.; Fangzheng, L.; Sanming, L. Platinum nanoparticle-doped multiwalled carbon-nanotube-modified glassy carbon electrode as a sensor for simultaneous determination of atenolol and propranolol in neutral solution. Ionics 2015, 21, 1129-1140.

[0073] [2] Shadjou, N.; Hasanzadeh, M.; Saghatforoush, L.; Mehdizadeh, R.; Jouyban, A. Electrochemical behavior of atenolol, carvedilol and propranolol on copper-oxide nanoparticles. Electrochim. Acta 2011, 58, 336 - 347.

[0074] [3] Kun, Z.; Yi, H.; Chengyun, Z.; Yue, Y.; Shuliang, Z.; Yuyang, Z. Electrochemical behavior of propranolol hydrochloride in neutral solution on platinum nanoparticles doped multi-walled carbon nanotubes modified glassy carbon electrode. Electrochim. Acta 2012, 80, 405 - 412.

[0075] [4] Kun, Z.; Shuai, Y.; Dongmei, T.; Yuyang, Z. Electrochemical behavior of propranolol hydrochloride in neutral solution on calixarene / multi-walled carbon nanotubes modified glassy carbon electrode. J. Electroanal. Chem. 2013, 709, 99 - 105.

[0076] [5] Li, H.-X.; Xu, X.-L.; Chen, H.; Zhang, S.; Kong, J.-L. Fabrication of molecularly imprinted electrochemical sensor for selective detection of propranolol hydrochloride. Chin. J. Anal. Chem. 2012, 40, 817 - 822.

[0077] [6]Alizadeh, T.; Allahyari, L. H. Highly-selective determination of carcinogenic derivative of propranolol by using a carbon paste electrode incorporated with nano-sized propranolol-imprinted polymer. Electrochim. Acta 2013, 111, 663-673.

[0078] [7]Gioia, D.; Casella, I. G. Pulsed electrodeposition of palladium nano-particles on coated multi-walled carbon nanotubes / nafion composite substrates: Electrocatalytic oxidation of hydrazine and propranolol in acid conditions. Sensor. Actuat. B-Chem. 2016, 237, 400-407.

[0079] [8]Wong, A.; Santos, A. M.; Silva, T. A.; Fatibello-Filho, O. Simultaneous determination of isoproterenol, acetaminophen, folic acid, propranolol and caffeine using a sensor platform based on carbon black, graphene oxide, copper nanoparticles and PEDOT:PSS. Talanta 2018, 183, 329-338.

[0080] [9]Santos, A.M.; Wong, A.; Fatibello-Filho, O. Simultaneous determination of salbutamol and propranolol in biological fluid samples using an electrochemical sensor based on functionalized-graphene, ionic liquid and silver nanoparticles. J. Electroanal. Chem. 2018, 824, 1 - 8.

[0081]

[10] Oliveira, G.G.; Azzi, D.C.; Vicentini, F.C.; Sartori, E.R.; Fatibello-Filho, O. Voltammetric determination of verapamil and propranolol using a glassy carbon electrode modified with functionalized multiwalled carbon nanotubes within a poly(allylamine hydrochloride) film. J. Electroanal. Chem. 2013, 708, 73 - 79.

[0082]

[11] Lourencao, B.C.; Silva, T.A.; Fatibello-Filho, O.; Swain, G.M. Voltammetric studies of propranolol and hydrochlorothiazide oxidation in standard and synthetic biological fluids using a nitrogen-containing tetrahedral amorphous carbon (ta-C:N) electrode. Electrochim. Acta 2014, 143, 398 - 406.

[0083] 3.3 Evaluation of stability, repeatability and interference studies.

[0084] To examine the stability of the electrochemical sensor, the prepared fDNA / AuNPs-MB-oxCNOs / GCE was stored at 4 °C for about two weeks. The change in the current response of the electrode stored in the same solution was 98.2% of the original current response. Compared with the current obtained two weeks ago, the current decreased by less than 3%, indicating that the stability of the modified electrode was acceptable. The repeatability was studied using three electrodes, and the relative standard deviations (RSDs) were 1.21% and 1.86% respectively, indicating that the electrochemical sensor had good repeatability.

[0085] The anti-interference ability of fDNA / AuNPs-MB-oxCNOs / GCE for the detection of PRO was tested in 0.1 M BR (pH 7.0) containing 100.0 μM PRO ( Figure 5 ). 500 μM Mg 2+ , Ca 2+ , SO4 2- , PO4 3- , AA, HAc, L-cys, and Glu were added respectively. The change in the signal of the electrochemical response was less than 5% and could be ignored. The results showed that fDNA / AuNPs-MB-oxCNOs / GCE had high selectivity and anti-interference ability for the detection of PRO and could be used for the determination of PRO in actual samples.

[0086] 3.4 Detection of PRO in real samples.

[0087] The sensor of the present invention was applied to determine PRO in biological fluid (urine and plasma) samples. No other electrochemical reactions were found in the potential range studied in plasma and synthetic urine samples. By adding a known volume of PRO standard solution to a given sample and analyzing it using the DPV technique, the detection of the PRO content and the recovery rate in the sample were studied, and the results are listed in Table 2. The recovery rates of the studied samples were good, with the recovery rates in plasma ranging from 92.5% to 105% and the recovery rates in synthetic urine samples ranging from 97.8% to 108%, indicating that the matrix had no obvious interference. Therefore, the proposed electrochemical method using DPV could sensitively, accurately, and reproducibly detect PRO in biological fluids.

[0088] Table 2 Detection results of PRO in human plasma and synthetic urine samples

[0089]

[0090]

[0091] 4. Conclusions.

[0092] The present invention successfully constructed a chemically modified glassy carbon electrode based on protamine DNA and carbon nanospheres loaded with gold nanoparticles and methylene blue for the detection of PRO. Due to the excellent electrochemical properties of the fDNA / AuNPs-MB-oxCNOs nanocomposite, such as well-defined redox peaks and a high electroactive surface area, a simple electrochemical sensor was constructed using the fDNA / Au NPs-MB-oxCNOs composite by differential pulse voltammetry for the detection of PRO. TEM and EDX analyses showed that MB and AuNPs were uniformly dispersed in the oxCNOs matrix. SEM analysis revealed the surface morphology of fDNA / AuNPs-MB-oxCNOs / GCE; the modified electrode achieved highly sensitive and selective detection of PRO with a linear range from 0.10 nM to 100 μM, good stability, and strong anti-interference performance. This method has been successfully used to determine PRO in biological fluid samples. The results of electrochemical performance tests showed that the fDNA / AuNPs-MB-oxCNOs nanocomposite is a powerful sensing material for constructing PRO electrochemical sensors.

[0093] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a nanocomposite material, characterized in that, It includes the following steps: Oxidize carbon nano-onions to obtain oxidized carbon nano-onions; mix the oxidized carbon nano-onions with methylene blue for the first time to obtain MB-oxCNOs nanomaterials; mix the MB-oxCNOs nanocomposites with gold nanoparticles for the second time to obtain AuNPs-MB-oxCNOs nanocomposites.

2. The preparation method of the nanocomposite material according to claim 1, characterized in that The oxidizing agent used for oxidation includes at least one of nitric acid and sulfuric acid; The mass ratio of the carbon nano-onions to the oxidizing agent is 30 - 36:14; The mass ratio of the oxidized carbon nano-onions to the methylene blue is 1:1 - 2; The mass ratio of the MB-oxCNOs nanocomposites to the gold nanoparticles is 1:0.1 - 2.

3. The preparation method of the nanocomposite material according to claim 1, wherein The parameters for oxidation are: temperature is 100°C - 110°C, time is 5h - 15h; The parameters for the first mixing are: ultrasonic frequency is 40KHz - 80KHz, time is 1h - 3h; The parameters for the second mixing are: stirring rate is 200rpm - 600rpm, time is 12h - 24h.

4. A nanocomposite material, characterized in that, The nanocomposite is prepared by the preparation method according to any one of claims 1 to 3.

5. The nanocomposite material according to claim 4, wherein The average particle size of the oxidized carbon nano-onions in the nanocomposite is 4.84nm; the average particle size of the gold nanoparticles in the nanocomposite is 14.5nm.

6. Glassy carbon modified electrode, characterized in that, The glassy carbon modified electrode includes a glassy carbon electrode and a carbon nanocomposite layer and a protamine DNA layer sequentially loaded on the glassy carbon electrode, and the material of the carbon nanocomposite layer includes the nanocomposite according to claim 4 or 5.

7. The preparation method of the glassy carbon modified electrode according to claim 6, characterized in that, It includes the following steps: first, modify the nanocomposite on the surface of the glassy carbon modified electrode to form a carbon nanocomposite layer; then modify protamine DNA on the carbon nanocomposite layer to form a protamine DNA layer to obtain a glassy carbon modified electrode.

8. The preparation method of the glassy carbon modified electrode according to claim 7, characterized in that, The mass ratio of the nanocomposite to the protamine DNA is 5:2 - 6.

9. Electrochemical sensor, characterized in that, The electrochemical sensor includes the glassy carbon modified electrode according to claim 6.

10. The electrochemical sensor according to claim 9, wherein, The electrochemical sensor further includes a reference electrode and a counter electrode.

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