Composite sol, electrode and preparation method and application thereof

Abstract

The invention belongs to the technical field of biomolecule detection, and particularly relates to composite sol, an electrode and a preparation method and application thereof. The composite sol comprises an hAu-Ru nano shell and poly (3-hexylthiophene); wherein the hAu-Ru nano shell contains Au and Ru elements and is in a hollow sphere shape, the diameter of the hAu-Ru nano shell ranges from 20 nm to 50 nm, and the shell thickness ranges from 5 nm to 10 nm. The biocompatibility and the stability of the CFME modified based on the composite sol, and the anti-interference capability, the selectivity, the sensitivity and the detection limit of ascorbic acid detection are obviously superior to those of the CFME modified based on the composite sol.

Description

Technical Field

[0001] The invention belongs to the technical field of biomolecule detection, and in particular relates to a composite sol, an electrode, and a preparation method and application thereof. Background Art

[0002] As a key water-soluble antioxidant in the brain, ascorbic acid (AA) plays a central role in maintaining redox balance, regulating neurotransmitter metabolism, and exerting neuroprotective effects. Its concentration dynamics (approximately 200±50μM under physiological conditions) are closely related to pathological processes such as cerebral edema and cerebral ischemia, making it an important biomarker for revealing the pathogenesis of neurodegenerative diseases. Therefore, achieving in situ, real-time, and highly sensitive monitoring of AA in the brain has important scientific value and clinical significance for studying its neuroprotective mechanisms and developing targeted treatment strategies. Currently, electrochemical sensors have become the mainstream technology for in vivo brain monitoring due to their high spatiotemporal resolution, fast response speed, and minimally invasive characteristics.

[0003] The complexity of the physiological environment in the brain (such as non-specific protein adsorption and the coexistence of multi-component neurochemical substances) poses severe challenges to AA detection: (1) Physiological interference factors: the presence of substances with similar electrochemical activity to AA, such as glucose, uric acid, and dopamine, in the brain can easily cause cross-interference; the adsorption of non-specific proteins on the electrode surface can lead to interface contamination and reduce detection stability; (2) Existing methods have limitations, such as the existing fluorescence / titration analysis method: it relies on exogenous markers, has poor biocompatibility, and cannot achieve in situ real-time monitoring; for example, for traditional electrochemical electrodes: bare carbon fiber microelectrodes (CFME) have insufficient sensitivity; electrodes modified with single metal nanomaterials (such as gold nanoparticles) have poor selectivity for glucose (AA / glucose selectivity ratio of about 1:3); polymer membrane (such as poly (3-hexylthiophene, P3HT)) modified electrodes improve biocompatibility, but have limited conductivity and catalytic activity; carbon nanotube composite electrodes have stability defects and biocompatibility risks caused by agglomeration. In other words, it is difficult for the above technologies to simultaneously meet the core requirements of "high selectivity, good biocompatibility, and in situ real-time monitoring".

[0004] Therefore, in view of the bottleneck that existing technologies cannot take into account selectivity, anti-pollution ability and biocompatibility, it is urgent to develop new electrode modification materials and sensing strategies to solve the interference problem and electrode failure risk of AA detection in the complex environment of the brain. Summary of the Invention

[0005] Based on this, the present invention uses hAu-Ru nanoshells to modify existing poly (3-hexylthiophene), and then forms a composite membrane on a carbon fiber microelectrode to achieve accurate detection of ascorbic acid, with good biocompatibility, high selectivity for ascorbic acid, and strong anti-pollution ability.

[0006] The present invention aims to break through the performance limitations of traditional single materials by constructing an electrochemical sensor modified with composite functional materials, achieve accurate monitoring of AA in the brain, and provide technical support for the study of neuropathological mechanisms.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] On the one hand, the present invention provides a composite sol comprising hAu-Ru nanoshells and poly (3-hexylthiophene); wherein the hAu-Ru nanoshells comprise Au and Ru elements, are hollow spherical, have a diameter of 20 nm to 50 nm, a shell thickness of 5 nm to 10 nm, and a specific surface area of ​​≥80 m 2 / g.

[0009] Preferably, the mass of Ru is 40% to 60% of the mass of Au; and / or the mass ratio of hAu-Ru nanoshell to poly (3-hexylthiophene) is (0.3 to 0.7):50.

[0010] Preferably, the preparation method of the hAu-Ru nanoshell comprises:

[0011] (1-1) mixing a reducing agent, a surface stabilizer, and a sacrificial template to obtain a mixed solution;

[0012] (2-1) Add Au to the mixture in sequence 3+ Ru 3+ Obtaining a reaction solution;

[0013] (3-1) The reaction solution was centrifuged to obtain hAu-Ru nanoshells.

[0014] More preferably, the preparation method of the hAu-Ru nanoshells satisfies one or more of the following conditions:

[0015] (a) the reducing agent is selected from one or more combinations of NaBH4, ascorbic acid, hydrazine hydrate or sodium citrate;

[0016] (b) a surface stabilizer selected from one or more of trisodium citrate, polyvinylpyrrolidone, sodium lauryl sulfate, or cetyltrimethylammonium bromide;

[0017] (c) The sacrificial template is selected from Co 2+ or Ag + ;

[0018] (d)Au 3+ from HAuCl4 or Au(NO3)3;

[0019] (e)Ru 3+ From RuCl3 or K2RuCl6.

[0020] Preferably, the composite sol further comprises a poly (3-hexylthiophene) oxidative dopant, which is used to promote oxidative cross-linking or doping between poly (3-hexylthiophene) chains.

[0021] Preferably, the poly (3-hexylthiophene) oxidative dopant is selected from one or more combinations of FeCl 3 , CuCl 2 or ammonium persulfate.

[0022] Another aspect of the present invention provides a method for preparing the composite sol, the method comprising:

[0023] (2-1) dissolving hAu-Ru nanoshells and poly (3-hexylthiophene) in an organic solvent to obtain a P3HT-hAu-Ru solution; dissolving poly (3-hexylthiophene) oxidized dopant in an organic solvent to obtain a poly (3-hexylthiophene) oxidized dopant solution;

[0024] (2-2) The poly (3-hexylthiophene) oxidized dopant solution is added to the P3HT-hAu-Ru solution to form a sol, namely a composite sol.

[0025] In another aspect, the present invention provides an electrode, which includes a carbon fiber microelectrode and a composite membrane, wherein the composite membrane is coated on the surface of the carbon fiber microelectrode; the composite membrane is obtained by drying the composite sol.

[0026] In another aspect, the present invention provides a three-electrode system, which includes the above-mentioned electrodes.

[0027] In another aspect, the present invention provides an application of the above-mentioned electrode or the above-mentioned three-electrode system, the application comprising:

[0028] (i) Use of the above-mentioned electrode in the preparation of a detection product for detecting ascorbic acid in the brain;

[0029] (ii) Use of the above electrode or the above three-electrode system in in vitro ascorbic acid detection.

[0030] The beneficial effects of the present invention include:

[0031] (1) The sensitivity of the carbon fiber microelectrode (CFME) modified with the composite sol provided by the present invention for detecting ascorbic acid can reach 120±15μA·mM-1·cm -2 , which is much higher than that of unmodified bare electrode (CFME) (20±5μA·mM-1·cm -2 ).

[0032] (2) The detection limit of ascorbic acid by the composite sol-modified carbon fiber microelectrode (CFME) provided by the present invention can be as low as 10 μM, which is much lower than that of the unmodified bare electrode (CFME) (50 μM).

[0033] (3) The ascorbic acid / glucose selectivity of the carbon fiber microelectrode (CFME) modified by the composite sol provided by the present invention can reach 1:20, which is much higher than that of the unmodified bare electrode (CFME) (1:3).

[0034] (4) When the anti-interference ability of the carbon fiber microelectrode (CFME) modified with the composite sol provided by the present invention is tested for glucose / uric acid / dopamine, the current fluctuation is less than 5%; while the current fluctuation of the unmodified bare electrode (CFME) is greater than 30%. The anti-interference ability of the carbon fiber microelectrode (CFME) modified with the composite sol provided by the present invention is significantly better than that of the unmodified bare electrode (CFME).

[0035] (5) The carbon fiber microelectrode (CFME) modified with the composite sol provided by the present invention has a fast response speed of 10 μM to 400 μM A at a potential of +0.3 V, a response time of <5 s, and a current fluctuation of <5% after 30 days, indicating good stability.

[0036] (6) The composite sol-modified carbon fiber microelectrode (CFME) provided by the present invention showed no inflammation and was tissue compatible 30 days after implantation in the body; while the unmodified bare electrode (CFME) showed moderate inflammation 30 days after implantation in the body; this indicates that the biocompatibility of the composite sol-modified carbon fiber microelectrode (CFME) provided by the present invention is better than that of the unmodified bare electrode (CFME). BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the STEM-EDS image of the hAu-Ru nanoshell prepared in Example 1;

[0038] Figure 2 This is a physical picture of the electrode (CFME / P3HT-hAu-Ru) prepared in Example 1;

[0039] Figure 3 CV graph of ascorbic acid tested using a three-electrode system consisting of electrodes prepared in Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0040] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0041] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0042] In the present invention, the “h” in “hAu-Ru nanoshell” stands for “hollow” and has no other specific meaning.

[0043] In a first aspect, an embodiment of the present invention provides a composite sol comprising hAu-Ru nanoshells and poly (3-hexylthiophene); wherein the hAu-Ru nanoshells comprise Au and Ru elements, are hollow spherical, have a diameter of 20nm-50nm, a shell thickness of 5nm-10nm, and a specific surface area of ​​≥80m 2 / g.

[0044] It should be noted that in the above-mentioned hAu-Ru nanoshell, Ru incorporation reduces the oxidation current of substances such as glucose, and the bimetallic synergistic effect of the hAu-Ru nanoshell separates the oxidation potential of ascorbic acid (AA) from interferences; and the hollow structure increases the specific surface area, and the high specific surface area and electron-rich core provide a large number of catalytic sites, thereby improving detection sensitivity; in addition, the inert surface of the hAu-Ru nanoshell reduces inflammatory response and improves biocompatibility; in addition, poly (3-hexylthiophene) can reduce protein adsorption and inflammatory response, improve anti-interference ability and biocompatibility, accelerate charge transfer, and also improve detection sensitivity.

[0045] It should also be noted that the diameter of the hAu-Ru nanoshell can be 20nm-50nm, such as 25nm, 30nm, 35nm, 40nm or 45nm; the shell thickness can be 5nm-10nm, such as 6nm, 7nm, 8nm or 9nm; the specific surface area ≥80m 2 / g, for example ≥100m 2 / g, etc.

[0046] In some specific examples, the mass of Ru is 40% to 60% of the mass of Au.

[0047] It should be noted that, in the hAu-Ru nanoshell of the present invention, the mass of Ru is 40% to 60% of the mass of Au, for example, 45%, 50%, 55% or 57%.

[0048] In some specific examples, the mass ratio of hAu-Ru nanoshell to poly (3-hexylthiophene) is (0.3-0.7):50.

[0049] It should be noted that the mass ratio of hAu-Ru nanoshell to poly (3-hexylthiophene) is (0.3-0.7):50, such as 0.4:50, 0.5:50, 0.6:50 or 0.7:50.

[0050] In some specific examples, the preparation method of the hAu-Ru nanoshell includes:

[0051] (1-1) mixing a reducing agent, a surface stabilizer, and a sacrificial template to obtain a mixed solution;

[0052] (2-1) Add Au to the mixture in sequence 3+ Ru 3+ Obtaining a reaction solution;

[0053] (3-1) The reaction solution was centrifuged to obtain hAu-Ru nanoshells.

[0054] It should be noted that in the preparation method of hAu-Ru nanoshell, the reaction needs to be carried out in a liquid environment, so the reducing agent, surface stabilizer, sacrificial template and Au 3+ Ru 3 They are preferably in the form of solutions.

[0055] In some specific examples, the preparation method of the hAu-Ru nanoshells satisfies one or more of the following conditions:

[0056] (a) The reducing agent is selected from one or more combinations of NaBH4, ascorbic acid, hydrazine hydrate or sodium citrate; specifically, the reducing agent in the present invention is to reduce the metal ions and Au in the sacrificial template to 3+ Ru 3+ The metal ions are reduced to nanoparticles, which can be selected from any reducing agent known in the art, such as the reducing agents listed above; NaBH4 is preferred in the present invention, which has a better effect in the present invention;

[0057] (b) a surface stabilizer selected from one or more of trisodium citrate, polyvinylpyrrolidone, sodium lauryl sulfate, or cetyltrimethylammonium bromide; specifically, the surface stabilizer in the present invention prevents agglomeration by adsorbing on the surface of the nanoparticles, thereby regulating the particle size and dispersibility. The surface stabilizer can be selected from those known in the art, such as the surface stabilizers listed above. Trisodium citrate is preferred in the present invention, as it has a better effect in the present invention;

[0058] (c) The sacrificial template is selected from Co 2+ or Ag +Specifically, the sacrificial template of the present invention can be selected from Co 2+ or Ag + , sacrificial templates, which form Co or Ag nanoparticles through reduction, and are subsequently coated with Au and Ru and then dissolved or removed to form a hollow structure;

[0059] (d)Au 3+ From HAuCl4 or Au(NO3)3; specifically, Au 3+ As is well known in the art, it can be derived from HAuCl4 or Au(NO3)3, preferably HAuCl4;

[0060] (e)Ru 3+ From RuCl3 or K2RuCl6; specifically, Ru 3+ As is well known in the art, it can be derived from RuCl3 or K2RuCl6, preferably RuCl3.

[0061] It should be noted that, among the conditions (a) to (e) in the above-mentioned method for preparing hAu-Ru nanoshells, it is preferred that two or more of the above conditions be satisfied, and it is more preferred that all of the above conditions be satisfied simultaneously.

[0062] In some specific examples, the composite sol further includes a poly (3-hexylthiophene) oxidative dopant, which is used to promote oxidative crosslinking or doping between poly (3-hexylthiophene) chains.

[0063] It should be noted that a poly(3-hexylthiophene) oxidative dopant can be added to the hAu-Ru nanoshells and poly(3-hexylthiophene) to promote oxidative crosslinking between poly(3-hexylthiophene) chains or to enhance interfacial interactions between the polymer and the nanoshells, thereby stabilizing the sol structure. Furthermore, it should be understood that if the composite sol does not contain the poly(3-hexylthiophene) oxidative dopant, other physical processes (e.g., ultrasound) are required to form the composite sol. Furthermore, the performance of the composite sol containing the poly(3-hexylthiophene) oxidative dopant is significantly superior to that of the composite sol without the poly(3-hexylthiophene) oxidative dopant.

[0064] In some specific examples, the poly (3-hexylthiophene) oxidative dopant is selected from one or more combinations of FeCl 3 , CuCl 2 or ammonium persulfate.

[0065] It should be noted that the poly (3-hexylthiophene) oxidative dopant in the present invention is well known in the art and can be any of those listed above, among which FeCl 3 is preferred, which has a better effect.

[0066] In a second aspect, an embodiment of the present invention provides a method for preparing the composite sol, the method comprising:

[0067] (2-1) dissolving hAu-Ru nanoshells and poly (3-hexylthiophene) in an organic solvent to obtain a P3HT-hAu-Ru solution; dissolving poly (3-hexylthiophene) oxidized dopant in an organic solvent to obtain a poly (3-hexylthiophene) oxidized dopant solution;

[0068] (2-2) The poly (3-hexylthiophene) oxidized dopant solution is added to the P3HT-hAu-Ru solution to form a sol, namely a composite sol.

[0069] It should be noted that, as mentioned above, when the above-mentioned composite sol does not contain poly (3-hexylthiophene) oxidized dopant, it is necessary to form the composite sol through other physical actions (such as ultrasound); when the above-mentioned composite sol contains poly (3-hexylthiophene) oxidized dopant, it can be prepared according to the above-mentioned preparation method, and the preparation method is simple and convenient.

[0070] In a third aspect, an embodiment of the present invention provides an electrode, comprising a carbon fiber microelectrode and a composite film, wherein the composite film is coated on the surface of the carbon fiber microelectrode; the composite film is obtained by drying the composite sol.

[0071] It should be noted that the composite sol is coated on the surface of the carbon fiber microelectrode (CFME), left to stand for 72 hours, and then washed and dried. The composite sol can form a composite film on the carbon fiber microelectrode (the specific process can be referred to Figure 1 ), that is, the electrode of the present invention can be prepared, that is, the electrode of the present invention is based on CFME, and the hAu-Ru nanoshell is evenly covered on the CFME surface; and the P3HT film (thickness of 50nm-100nm) formed by poly 3-hexylthiophene (P3HT film) wraps the hAu-Ru nanoshell to form a composite conductive network; and the P3HT film can also block large molecular interferents (such as glucose and protein) through pore size screening, while allowing AA small molecules to pass through; specifically, the pore size of the P3HT membrane is 2nm~5nm, while the diameter of the AA small molecule is 0.6nm, and the diameter of the glucose molecule is about 0.8nm, that is, the size of the glucose molecule is large and difficult to penetrate, thereby achieving selective filtration.

[0072] Specifically, the composite membrane formed by the composite sol of the present invention is composed of poly (3-hexylthiophene) (P3HT) and hAu-Ru nanoshells. Its nominal pore size is 2nm-5nm. However, as a conjugated polymer, the actual effective channels formed by the accumulation of P3HT molecular chains may produce a "dynamic pore size" due to chain entanglement, crosslinking, or the embedding of nanoshells. This means that the actual permeable channel size is smaller than the nominal pore size, and the hydrophobic / hydrophilic properties of the polymer may form a selective barrier inside the channel. This structure makes it difficult for molecules with a size smaller than the nominal pore size (such as glucose 0.8nm) to penetrate due to steric hindrance or channel morphology.

[0073] Furthermore, the addition of Ru to the hAu-Ru nanoshell enables selectivity for AA without the aid of enzymes or membranes, surpassing interferents such as glucose. Combined with the design of the composite membrane, the hydrophobic chains of P3HT may interact more strongly (e.g., hydrogen bonds and van der Waals forces) with glucose molecules (which contain multiple hydroxyl groups and are highly polar), resulting in glucose molecules being adsorbed and retained by the membrane surface or the inner walls of the channels, making them difficult to penetrate. Meanwhile, the molecular structure of AA (relatively weak in polarity and reducing properties) interacts less strongly with the membrane material, allowing it to pass through the channels more easily, thus achieving selective filtration.

[0074] In addition, the catalytic specificity of the hAu-Ru nanoshell and the screening effect of the P3HT membrane work together as follows: the hAu-Ru nanoshell has a higher catalytic activity for AA, while the P3HT membrane further blocks interferences such as glucose through the above-mentioned structure and chemical effects. Even though the two molecules have similar sizes, they can still achieve selective detection of AA through multiple mechanisms.

[0075] In a fourth aspect, an embodiment of the present invention provides a three-electrode system, which includes the above-mentioned electrodes.

[0076] It should be noted that the electrode prepared in the present invention can be used as a working electrode to form a three-electrode system to detect ascorbic acid; wherein the reference electrode and the counter electrode of the three-electrode system are well known in the art, for example, the reference electrode can be Ag / AgCl and the counter electrode can be Pt.

[0077] In a fifth aspect, an embodiment of the present invention provides a use of the above-mentioned electrode in preparing a detection product for detecting ascorbic acid in the brain.

[0078] It should be noted that the electrode of the present invention has excellent anti-interference ability and selectivity when detecting ascorbic acid, and can be prepared into a detection product for detecting ascorbic acid in the brain for the detection and monitoring of ascorbic acid in the brain.

[0079] In a sixth aspect, an embodiment of the present invention provides a use of the above-mentioned electrode or the above-mentioned three-electrode system in in vitro ascorbic acid detection.

[0080] It should be noted that the electrodes of the present invention can not only detect ascorbic acid in the brain, but also detect ascorbic acid in vitro; at the same time, the prepared three-electrode system can also detect ascorbic acid in vitro.

[0081] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0082] In the following example, the pretreatment process of CFME (carbon fiber microelectrode) includes: ultrasonic cleaning of carbon fiber with acetone, ethanol, and ultrapure water in sequence to remove organic matter and debris; possible immersion in dilute acid (such as HNO3) or dilute alkali (such as KOH) solution to activate the surface and introduce oxygen-containing functional groups (such as hydroxyl and carboxyl) to enhance conductivity and biocompatibility; adhering the carbon fiber microelectrode to the copper wire with silver glue and drying; then encapsulating with a glass capillary and cutting the carbon fiber tip.

[0083] In the following example, the preparation process of CFME (carbon fiber microelectrode) includes: pre-treating (cleaning and activating) commercially available carbon fiber materials for subsequent modification; encapsulating them in a flexible substrate (implicitly containing glass / polymer) to expose the working end; and cutting the carbon fiber to the size of a microelectrode, which is about 1 cm.

[0084] Preparation Example

[0085] Example 1

[0086] (1) Preparation of hAu-Ru nanoshells

[0087] (1-1) Under nitrogen atmosphere, heat 135 mL of deionized water to 60°C and stir vigorously for more than 30 minutes;

[0088] (1-2) 5 mL of NaBH4 aqueous solution (concentration 0.24 M) and 2.5 mL of trisodium citrate aqueous solution (0.12 M) were added sequentially and stirred for 5 minutes;

[0089] (1-3) Add 5 mL of CoCl2 aqueous solution (concentration 75 mM) dropwise and stir for 15 minutes;

[0090] (1-4) First, add 5 mL of HAuCl4 aqueous solution (concentration is 30 mM) dropwise, then add 5 mL of RuCl3 aqueous solution (concentration is 30 mM) dropwise after 5 minutes, and continue to stand for 15 minutes;

[0091] (1-5) The obtained sample was washed with water for at least 5 times and collected by centrifugation (centrifugal speed 8000 rpm, time 10 minutes) to obtain hAu-Ru nanoshells.

[0092] (2) Preparation of P3HT-hAu-Ru sol

[0093] (2-1) 0.5 mg of P3HT (poly (3-hexylthiophene)) and 50 mg of hAu-Ru nanoshells were added to 10 mL of chloroform and ultrasonically dispersed for 1 h (ultrasonic power 300 W, temperature 25°C) to obtain a P3HT-hAu-Ru chloroform solution;

[0094] (2-2) Add 1.9 g of anhydrous FeCl3 to 20 mL of chloroform and stir for 1 hour (500 rpm) to obtain a FeCl3 chloroform solution;

[0095] (2-3) Slowly add the FeCl3 chloroform solution to the P3HT-hAu-Ru chloroform solution and continue stirring for 2 hours to form a P3HT-hAu-Ru sol.

[0096] (3) Electrode (CFME / P3HT-hAu-Ru) preparation

[0097] (3-1) The pretreated CFME (carbon fiber microelectrode) (diameter of 10 μm) was inserted into the P3HT-hAu-Ru sol prepared in step (2) above and allowed to stand for 72 h;

[0098] (3-2) After the rest period, the CFME was removed and washed (the electrode was repeatedly rinsed until the effluent was colorless), and then dried naturally (dried at room temperature to retain the flexibility and structural integrity of the membrane) to obtain the treated carbon fiber microelectrode, i.e., the electrode (CFME / P3HT-hAu-Ru).

[0099] Example 2

[0100] Example 2 is substantially the same as Example 1, except that CuCl2 is used instead of FeCl3 in step (2) of Example 2, and the rest is the same as Example 1.

[0101] Example 3

[0102] Example 2 is substantially the same as Example 1, except that the amount of P3HT used in step (2) of Example 3 is 0.3 mg, and the rest is the same as Example 1.

[0103] Example 4

[0104] Example 4 is substantially the same as Example 1, except that the amount of P3HT used in step (2) of Example 4 is 0.7 mg, and the rest is the same as Example 1.

[0105] Comparative Example 1

[0106] CFME (carbon fiber microelectrode) was used as comparative example 1.

[0107] Comparative Example 2

[0108] (1) Preparation of hAu nanoshells

[0109] (1-1) Under nitrogen atmosphere, 135 mL of deionized water was vigorously stirred for more than 30 minutes;

[0110] (1-2) 5 mL of NaBH4 aqueous solution (concentration 0.24 M) and 2.5 mL of trisodium citrate aqueous solution (0.12 M) were added sequentially and stirred for 5 minutes;

[0111] (1-3) Add 5 mL of CoCl2 aqueous solution (concentration 75 mM) dropwise and stir for 15 minutes;

[0112] (1-4) Then, 5 mL of HAuCl4 aqueous solution (concentration of 30 mM) was added dropwise and allowed to stand for 15 minutes;

[0113] (1-5) The obtained sample was washed with water at least 5 times and collected by centrifugation (centrifugal speed 8000 rpm, time 10 minutes) to obtain hAu nanoshells.

[0114] (2) Preparation of hAu chloroform solution

[0115] (2-1) 50 mg of hAu nanoshells were added to 10 mL of chloroform and ultrasonically dispersed for 1 hour (ultrasonic power of 300 W, temperature of 25° C.) to obtain a hAu chloroform solution.

[0116] (3) Electrode (CFME / P3HT-hAu) preparation

[0117] (3-1) The pretreated CFME (carbon fiber microelectrode) (diameter 10 μm) was inserted into the hAu chloroform solution prepared in step (2) above and allowed to stand for 72 h;

[0118] (3-2) After the rest period, the CFME was removed and washed (the electrode was repeatedly rinsed until the effluent was colorless), and then dried naturally (dried at room temperature to retain the flexibility and structural integrity of the membrane) to obtain the treated carbon fiber microelectrode, i.e., the electrode (CFME / hAu).

[0119] Comparative Example 3

[0120] (1) Preparation of P3HT sol

[0121] (1-1) 0.5 mg of P3HT (poly (3-hexylthiophene)) was added to 10 mL of chloroform and ultrasonically dispersed for 1 hour (ultrasonic power 300 W, temperature 25°C) to obtain a P3HT chloroform solution.

[0122] (1-2) Add 1.9 g of anhydrous FeCl3 to 20 mL of chloroform and stir for 1 hour (500 rpm) to obtain a FeCl3 chloroform solution;

[0123] (1-3) Slowly add FeCl3 chloroform solution to P3HT chloroform solution and continue stirring for 2 hours to form P3HT sol.

[0124] (2) Electrode (CFME / P3HT) preparation

[0125] (2-1) The pretreated CFME (carbon fiber microelectrode) (diameter of 10 μm) was inserted into the P3HT sol prepared in step (2) above and allowed to stand for 72 h;

[0126] (2-2) After the rest period, the CFME was removed and washed (the electrode was repeatedly rinsed until the effluent was colorless), and then dried naturally (dried at room temperature to retain the flexibility and structural integrity of the membrane) to obtain the treated carbon fiber microelectrode, i.e., the electrode (CFME / P3HT).

[0127] Characterization data

[0128] STEM-EDS observation of the hAu-Ru nanoshell prepared in Example 1 showed the following results: Figure 1 As shown in the figure, the results show that Au (blue) and Ru (red) elements are evenly distributed, confirming that the hollow nanoshell is successfully prepared. In addition, HR-TEM observation of the hAu-Ru nanoshell structure prepared in Example 1 shows that it is a hollow structure with a thickness of about 10nm-20nm; and the specific surface area test (BET) shows 102m 2 / g.

[0129] In addition, the actual photo of CFME / P3HT-hAu-Ru prepared in Example 1 is as follows: Figure 1 Among them, hAu-Ru nanoshell (thickness of 15nm) is evenly wrapped on CFME, and the hAu-Ru nanoshell is wrapped with P3HT film (thickness of 83nm), forming a core-shell-membrane three-layer structure.

[0130] Performance Testing

[0131] (1) Sensitivity test

[0132] In the test of the present invention, the sensitivity test method is as follows:

[0133] (1) Ascorbic acid (AA) was added to artificial cerebrospinal fluid (purchased from Amazon, containing 126 mM NaCl, 2.4 mM KCl, 0.5 mM KH2PO4, 0.85 mM MgCl2, 27.5 mM NaHCO3, 0.5 mM Na2SO4, and 1.1 mM CaCl2, the same below) to obtain ascorbic acid, so that the concentrations of ascorbic acid in the artificial cerebrospinal fluid were 0, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, and 4 mM, respectively;

[0134] (2) Using a three-electrode system (working electrode, reference electrode (Ag / AgCl), counter electrode (Pt)), cyclic voltammetry (CV) was performed to sequentially test ascorbic acid solutions with different ascorbic acid concentrations, and the oxidation peak current was recorded. The scanning voltage range of the cyclic voltammetry (CV) was -0.1 V to +0.7 V, and the scanning rate was 50 to 200 mV / s.

[0135] (3) Sensitivity = ΔI / ΔC, where ΔI is the change in oxidation peak current (μA) and ΔC is the change in ascorbic acid concentration (mM).

[0136] The electrodes prepared in Example 1 and Comparative Example 1 were used as working electrodes in a three-electrode system to test their sensitivity to ascorbic acid. Figure 3 As shown ( Figure 3 In the figure, two curves of the same color are parallel experiments); in addition, the test results of the embodiment and the comparative example are shown in Table 1 below.

[0137] Table 1 Sensitivity of electrodes prepared in Examples / Comparative Examples to ascorbic acid

[0138] Examples / Comparative Examples <![CDATA[Sensitivity (μA·mM-1·cm -2 )]]> Example 1 120±15 Example 2 113±12 Example 3 110±13 Example 4 112±14 Comparative Example 1 20±5 Comparative Example 2 80±10 Comparative Example 3 50±8

[0139] Depend on Figure 3 As can be seen from Table 1 above, the sensitivity of the electrodes prepared in the examples is significantly better than that of the comparative examples; among them, the electrode prepared in Example 1 has a linear response to the oxidation peak current of 0-4mMAA, and the sensitivity is 120±15μA·mM-1·cm -2 , the sensitivity is the best; the electrode in comparative example 1 (bare CFME) is 20±5μA·mM-1·cm -2 , the worst sensitivity.

[0140] (2) Detection limit test

[0141] In the test of the present invention, the test method of the detection limit is as follows:

[0142] (1) Using a three-electrode system (working electrode, reference electrode (Ag / AgCl), counter electrode (Pt)), the electrochemical response of artificial cerebrospinal fluid was continuously tested 20 times by cyclic voltammetry (CV). The blank current was recorded each time, and the standard deviation of the blank current was calculated, denoted as σ.

[0143] (2) adding different amounts of ascorbic acid (AA) to the artificial cerebrospinal fluid to obtain ascorbic acid, such that the concentrations of ascorbic acid in the artificial cerebrospinal fluid are 0, 2 mM, and 4 mM, respectively;

[0144] (3) Using a three-electrode system (working electrode, reference electrode (Ag / AgCl), counter electrode (Pt)), cyclic voltammetry (CV) was performed to sequentially test ascorbic acid solutions with different ascorbic acid concentrations, and the oxidation peak current was recorded. The scanning voltage range of the cyclic voltammetry (CV) was -0.1 V to +0.7 V, and the scanning rate was 50 mV / s to 200 mV / s.

[0145] (4) With ascorbic acid concentration (C, μM) as the horizontal axis, the oxidation peak current (I p , μA) is the vertical axis, and I p -C standard curve; I p The slope of the -C standard curve is recorded as s;

[0146] (5) Calculate the detection limit based on the detection limit (LOD) = 3σ / s.

[0147] The electrodes prepared in the embodiment and the comparative example were respectively used as working electrodes of a three-electrode system to test their detection limits for ascorbic acid. The results are shown in Table 2 below.

[0148] Table 2 Detection limits of ascorbic acid for electrodes prepared in Examples / Comparative Examples

[0149] Examples / Comparative Examples Detection limit (μM) Example 1 10 Example 2 15 Example 3 13 Example 4 12 Comparative Example 1 50 Comparative Example 2 20 Comparative Example 3 30

[0150] It can be seen from Table 2 above that the detection limits of the electrodes prepared in the examples are significantly lower than those in the comparative examples; among them, the detection limit of the electrode prepared in Example 1 for ascorbic acid can be as low as 10 μM, which is the lowest detection limit; the electrode in comparative example 1 (bare CFME) is 50 μM, which has the highest detection limit.

[0151] (III) Ascorbic acid / glucose selectivity test

[0152] In the test of the present invention, the test method for ascorbic acid / glucose selectivity is as follows:

[0153] (1) Artificial cerebrospinal fluid was divided into several portions, and ascorbic acid (AA) was added to each portion of artificial cerebrospinal fluid so that the concentration of ascorbic acid in the artificial cerebrospinal fluid was 10 μM; then, different amounts of glucose were added to each portion of artificial cerebrospinal fluid containing ascorbic acid to obtain test solutions, so that the concentrations of glucose were 30 μM, 50 μM, 100 μM, 150 μM, 170 μM, 180 μM, and 200 μM, respectively;

[0154] (2) Connect the working electrode, reference electrode, and counter electrode to the WE, RE, and CE interfaces of an electrochemical workstation (Haichenhua CHI660F), respectively; select cyclic voltammetry (CV) for testing; wherein the scanning voltage range in cyclic voltammetry (CV) is -0.1 V to +0.7 V, and the scanning rate is 50 mV / s to 200 mV / s;

[0155] (3) Add 5 mL of the test solution to a 10 mL electrolytic cell and insert three electrodes, ensuring that the electrode tips are immersed and do not touch each other. After deoxygenation, record the CV curve. The selectivity for ascorbic acid is determined based on the electrode response currents to ascorbic acid and glucose.

[0156] The electrodes prepared in the embodiment and the comparative example were used as working electrodes, respectively, and their selectivity to ascorbic acid / glucose was tested according to the above test method. The results are shown in Table 3 below.

[0157] Table 3 Selectivity of electrodes prepared in Examples / Comparative Examples for ascorbic acid / glucose

[0158] Examples / Comparative Examples Ascorbic acid / glucose selectivity Example 1 1:20 Example 2 1:15 Example 3 1:18 Example 4 1:17 Comparative Example 1 1:3 Comparative Example 2 1:5 Comparative Example 3 1:10

[0159] It can be seen from Table 3 above that the ascorbic acid / glucose selectivity of the electrodes prepared in the examples are significantly better than that of the comparative examples; among them, the response current of the electrode prepared in Example 1 to ascorbic acid is 200 nA, and the response current to glucose is only 10 nA, with a response ratio of 20:1, that is, the selectivity ratio is 1:20 (ascorbic acid / glucose); that is, when the glucose concentration is 20 times that of ascorbic acid, the electrode can still preferentially detect ascorbic acid; in addition, the electrode in Comparative Example 1 (bare CFME) has the lowest ascorbic acid / glucose selectivity, which is 1:3.

[0160] (3) Anti-interference test

[0161] (1) Test system preparation

[0162] A three-electrode system was used: the working electrode was the electrode prepared in the examples and comparative examples, the reference electrode was a standard Ag / AgCl electrode, and the counter electrode was a Pt electrode; the test medium was artificial cerebrospinal fluid; the detection instrument was a CHI630e electrochemical workstation, and the detection method was cyclic voltammetry (CV).

[0163] (2) Reference signal

[0164] Ascorbic acid (AA) was added to artificial cerebrospinal fluid to a final concentration of 200 μM, a physiological concentration. The electrochemical signal of AA on the sensor was measured using the above-mentioned test system. The current response value was recorded as the reference signal (I0). The measurement was repeated three times and the average value was taken to reduce the error.

[0165] (3) Interference addition and signal detection

[0166] Glucose (5 mM), uric acid (UA, 0.1 mM), dopamine (DA, 0.02 mM), 4-acetaminophen (AP, 0.1 mM) and nicotinamide adenine dinucleotide (NADH, 0.1 mM) were added separately to the above-mentioned artificial cerebrospinal fluid containing ascorbic acid. After each addition of an interfering substance, the system was stirred to mix evenly. After stabilization for 5 minutes, the electrochemical signal (I1) of AA was measured and recorded using the above-mentioned test system. The measurement was repeated 3 times for each interfering substance, and the average value was taken.

[0167] (4) Data calculation and anti-interference evaluation

[0168] Calculate the relative deviation of the AA signal after adding each interferent: relative deviation = |(I1-I0) / I0| × 100%;

[0169] If the relative deviation is ≤10%, it is considered that the electrode's anti-interference ability to the interference meets the standard; otherwise, the anti-interference ability is insufficient.

[0170] The electrodes prepared in the examples and comparative examples were tested for interference with ascorbic acid (AA) using glucose (5 mM), uric acid (UA, 0.1 mM), dopamine (DA, 0.02 mM), 4-acetaminophen (AP, 0.1 mM), and nicotinamide adenine dinucleotide (NADH, 0.1 mM), respectively, according to the above steps. The results are shown in Table 4 below.

[0171] Table 4 Anti-interference test results of electrodes prepared in Examples / Comparative Examples

[0172]

[0173] It can be seen from Table 4 above that the relative deviations of the electrodes prepared in Example under different interferences are all less than 10%, meeting the anti-interference standard; the relative deviations of the electrodes prepared in Example under different interferences are all greater than 10%, and are much higher than Example 1, and do not meet the anti-interference standard.

[0174] (4) Biocompatibility testing

[0175] The electrodes prepared in the examples and comparative examples were subjected to biocompatibility tests, as follows:

[0176] (1) Mice were used as experimental subjects, and electrodes were implanted into their brains using a stereotaxic apparatus after anesthesia.

[0177] (2) 30 days after electrode implantation, the experimental mice underwent surgery to remove the implanted electrodes and surrounding brain tissue;

[0178] (3) The brain tissue around the electrodes was processed (such as fixation and slicing) and CD68 was observed by immunohistochemistry and other methods.+ Activated macrophages (marker of acute inflammatory response) and CD206 + The distribution of M2 macrophages (markers of tissue repair and anti-inflammatory response) was analyzed to calculate the proportion of the two types of cells, thereby evaluating the degree of tissue inflammation and determining the biocompatibility of the electrode.

[0179] The test results are shown in Table 5 below.

[0180] Table 5 Biocompatibility test results of electrodes prepared in Examples / Comparative Examples

[0181] Examples / Comparative Examples Biocompatibility (30 days after implantation) Example 1 No inflammation, tissue compatible Comparative Example 1 Moderate inflammation Comparative Example 2 Mild inflammation Comparative Example 3 No obvious inflammation

[0182] It can be seen from Table 5 above that the electrode prepared in Example 1 of the present invention had no inflammation 30 days after implantation, indicating excellent biocompatibility; the electrode prepared in Comparative Example 3 had no obvious inflammation 30 days after implantation, indicating good biocompatibility; while the electrode in Comparative Example 1 produced moderate inflammation, and the electrode in Comparative Example 2 produced mild inflammation, indicating that poly (3-hexylthiophene) had excellent biocompatibility and was better than CFME / hAu in Comparative Example 2.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A composite sol, characterized in that: It includes hAu-Ru nanoshell and poly 3-hexylthiophene; wherein, the hAu-Ru nanoshell contains Au and Ru elements, is hollow spherical, has a diameter of 20nm to 50nm, a shell thickness of 5nm to 10nm, and a specific surface area of ​​≥80m 2 / g.

2. The composite sol according to claim 1, characterized in that The mass of Ru is 40% to 60% of the mass of Au; and / or The mass ratio of hAu-Ru nanoshell to poly (3-hexylthiophene) is (0.3-0.7):

50.

3. The composite sol according to claim 1 or 2, characterized in that The preparation method of hAu-Ru nanoshells includes: (1-1) mixing a reducing agent, a surface stabilizer, and a sacrificial template to obtain a mixed solution; (2-1) Add Au to the mixture in sequence 3+ Ru 3+ Obtaining a reaction solution; (3-1) The reaction solution was centrifuged to obtain hAu-Ru nanoshells.

4. The composite sol according to claim 3, characterized in that The preparation method of hAu-Ru nanoshells meets one or more of the following conditions: (a) the reducing agent is selected from one or more combinations of NaBH4, ascorbic acid, hydrazine hydrate or sodium citrate; (b) a surface stabilizer selected from one or more of trisodium citrate, polyvinylpyrrolidone, sodium lauryl sulfate, or cetyltrimethylammonium bromide; (c) The sacrificial template is selected from Co 2+ or Ag + ; (d)Au 3+ from HAuCl4 or Au(NO3)3; (e)Ru 3+ From RuCl3 or K2RuCl6.

5. The composite sol according to any one of claims 1 to 4, characterized in that The composite sol further comprises a poly (3-hexylthiophene) oxidative dopant, which is used to promote oxidative cross-linking or doping between poly (3-hexylthiophene) chains.

6. The composite sol according to claim 5, characterized in that The poly (3-hexylthiophene) oxidative dopant is selected from one or more combinations of FeCl 3 , CuCl 2 or ammonium persulfate.

7. The method for preparing the composite sol according to claim 5 or 6, characterized in that: The preparation method of the composite sol comprises: (2-1) dissolving hAu-Ru nanoshells and poly (3-hexylthiophene) in an organic solvent to obtain a P3HT-hAu-Ru solution; dissolving poly (3-hexylthiophene) oxidized dopant in an organic solvent to obtain a poly (3-hexylthiophene) oxidized dopant solution; (2-2) The poly (3-hexylthiophene) oxidized dopant solution is added to the P3HT-hAu-Ru solution to form a sol, namely a composite sol.

8. An electrode, characterized in that The invention comprises a carbon fiber microelectrode and a composite membrane, wherein the composite membrane is coated on the surface of the carbon fiber microelectrode; the composite membrane is obtained by drying the composite sol according to any one of claims 1 to 6.

9. A three-electrode system, characterized in that Comprising the electrode according to claim 8.

10. Use of the electrode according to claim 8 or the three-electrode system according to claim 9, comprising: (i) Use of the electrode according to claim 8 in the preparation of a detection product for detecting ascorbic acid in the brain; (ii) Use of the electrode according to claim 8 or the three-electrode system according to claim 9 in in vitro ascorbic acid detection.

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