阅读说明：本技术 一种纳米复合物电化学传感器、构建方法及其在对于葡萄糖的电化学发光法检测中的应用 (Nanocomposite electrochemical sensor, construction method and application of nanocomposite electrochemical sensor in electrochemical luminescence detection of glucose ) 是由 李小荣 仲慧 张安然 程志鹏 盛振环 殷竟洲 张载超 包转丽 刘易鑫 陈琪 于 2020-04-07 设计创作，主要内容包括：本发明提供的电化学发光(ECL)传感器是用羧基化石墨烯负载的Au@Ag核壳纳米粒子开发的。基于ECL的实验结果,我们发现COOH-G/@Au@Ag纳米复合材料对葡萄糖具有良好的电催化能力,因此构建了葡萄糖电化学发光(ECL)传感器。当葡萄糖浓度范围在0.005至1500μM之间变化时,ECL强度与葡萄糖浓度之间具有良好的线性响应,检出限为0.02μM。此外,开发的葡萄糖ECL传感器不仅具有良好的稳定性,可重复性和灵敏度,还可以成功地用于人体血清样品中的葡萄糖的监测。(The electrochemical luminescence (ECL) sensor provided by the invention is developed by using the Au @ Ag nuclear shell nano-particles loaded by the carboxylated graphene. Based on the ECL experimental result, the COOH-G/@ Au @ Ag nano composite material is found to have good electrocatalytic capacity on glucose, so that the glucose electrochemical luminescence (ECL) sensor is constructed. When the glucose concentration range is changed between 0.005 and 1500 mu M, a good linear response is obtained between the ECL intensity and the glucose concentration, and the detection limit is 0.02 mu M. In addition, the developed glucose ECL sensor not only has good stability, repeatability and sensitivity, but also can be successfully used for monitoring glucose in human serum samples.)

1. A nanocomposite electrochemical sensor is characterized in that the nanocomposite electrochemical sensor is an electrode loaded with a COOH-G/Au @ Ag material.

2. The nanocomposite electrochemical sensor according to claim 1, wherein the COOH-G/Au @ Ag material is a nanoparticle coated with gold and silver on the surface of the carboxylated graphene in sequence.

3. The method of preparing a nanocomposite electrochemical sensor of claim 1, comprising the steps of:

polishing the surface of the glassy carbon electrode;

and preparing a suspension of the COOH-G/Au @ Ag material, adding the suspension to the surface of the carbon breaking electrode, and drying to obtain the carbon breaking electrode.

4. The method of claim 1, wherein the suspension of the COOH-G/Au @ Ag material has a concentration of 1mg mL in one embodiment^-1。

5. Use of the nanocomposite electrochemical sensor of claim 1 for the detection of a solution containing glucose.

6. The use of claim 5, wherein in one embodiment, the detection is by electrochemiluminescence.

7. The use of claim 5, wherein in one embodiment, the solution comprises 1mM glucose.

8. Use according to claim 5, wherein, in one embodiment, the scan rate in the assay is 25-300 nmvs^-1。

9. Use according to claim 5, wherein in one embodiment the luminol content in the assay is controlled between 0.1 and 0.6mM, preferably between 0.1 and 0.4 mM.

Technical Field

The invention relates to a nanocomposite electrochemical sensor, a construction method and application thereof in electrochemical luminescence detection of glucose, and belongs to the technical field of electrochemistry.

Background

Graphene is a two-dimensional thin-layer structure formed by closely-packed single-layer carbon atoms, and has a high electron migration speed and a large specific surface area (the theoretical specific surface area is about 2600 m)²g^-1) And excellent chemical and mechanical properties [ 1]]And thus has attracted considerable attention in the research fields of electrochemical sensors and biosensors. It is a two-dimensional material with a cellular lattice [ 2]]. Due to its high conductivity and large specific surface area, it has very high biomolecule load, which makes it widely explored and applied in electrochemical biosensing research [3]. However, simple graphene has an inert surface, which means high chemical stability and relatively weak interaction with the medium. In addition, graphene sheets have strong van der Waals and electrostatic forces between them, and thus aggregation is easily generated [4]. It is difficult to disperse in water and common organic solvents and has strong hydrophobicity, which not only brings certain difficulty to further research of graphene, but also limits the application and development of graphene in many fields [5 ]]. Therefore, researchers have functionalized graphene modifications to impart new functionalities to them by introducing specific functional groups, while maximizing their usefulnessRetains the excellent performance of graphene per se [6 ]]. Carboxylated graphene (COOH-G) has a large number of polar oxygen groups, and thus high chemical and hydrophilic activities are generated between layers of the COOH-G, which allows the COOH-G to have a monoatomic layer structure that can be laterally stretched. In addition, the increased hydrophilicity facilitates electrochemical modification, which makes COOH-G useful as a support material for nanoparticles and directly for electrochemical applications [7 ]]。

Noble metal nanocrystalline materials have unique and novel optical, electrical, magnetic and catalytic properties and are therefore highly attractive and of increasing interest [8 ]. By controlling the reaction conditions, the size, the form and the structure of the nano-particles can be adjusted to improve the surface catalytic performance, and the nano-particles of the noble metal can be used in the fields of catalysts, sensors, medical detection, imaging and the like. Single noble metal nano materials such as gold (Au) 9, platinum (Pt) 10, palladium (Pd) 11 and silver (Ag) 12 have strong conductivity and high catalytic performance 13. The double noble metal nano structure fully utilizes the electron coupling effect and the synergistic catalytic performance, so that the nano structure has increased specific surface area and enhanced sensitivity while maintaining effective recognition capability. However, since the noble metal nanoparticles are prone to form aggregates during the preparation process due to the large surface energy, how to improve the stability is a major challenge. The monolayer atomic structure of COOH-G reduces the surface free energy in the presence of wrinkles. In addition, the carboxyl function increases the interlayer distance and thus improves the stability of COOH-G. With the reduction of noble metal nanoparticles on the COOH-G groove-shaped surface, the composite material not only retains the effective catalysis and conductivity of the composite material, but also greatly improves the overall stability, and has wide application prospect in the sensing field.

It is well known that serum testing has become a routine method for monitoring and diagnosing various medical conditions [14]. Glucose is the main component of carbohydrates in plants and animals, and is one of the essential substances in daily life [15]. The glucose level in blood has been used to diagnose diabetes or hypoglycemia. In addition to requiring blood glucose monitoring in diabetic patients, controlling blood glucose levels is also important in non-diabetic emergency patientsTo [16-18 ]]. Glucose is detected by a number of methods, including 3, 5-dinitrosalicylic acid (DNS), High Performance Liquid Chromatography (HPLC), iodometry, and electrochemical methods. Among these methods, Electrochemiluminescence (ECL) is a combined product of electrochemistry and chemiluminescence, showing high sensitivity, low background signal, good selectivity and spatial controllability. ECL biosensor as an effective analysis tool can accurately monitor the glucose content in actual sample (mainly blood) [19-21 ]]. Detection of target molecules with electrochemical sensors is an efficient and simple detection method. As a combination between electrochemical and spectroscopic techniques, ECL has unique advantages over other detection systems, such as chemiluminescence and fluorescence [22-24 ]]. In ECL experiments, intermediates are formed on electrodes, and during the reaction, these intermediates undergo high-energy electron transfer reactions, forming excited states and emitting light [25-26 ]]. In particular, luminol-based biosensors play an increasingly important role in bioassays. This is because the important bio-metabolic molecule H₂O₂Has lower oxidation potential and higher luminous quantum yield, thereby greatly improving the emission of luminol [27-30 ]]. ECL electrochemical sensors play an increasingly important role in the field of disease diagnosis and therapy.

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Disclosure of Invention

The invention adopts a two-step reduction method to synthesize COOH-G loaded with Au @ Ag core-shell nanoparticles. The nanocomposite combines the excellent electrocatalytic activity and conductivity of AuNPs and AgNPs with the large specific surface area of graphene materials, so that the compound has better electronic conductivity, and further improves the electron transfer rate between the electrode surface and an analyte in a supporting electrolyte solution. On the basis, an enzyme-free ECL biosensor with a COOH-G/Au @ Ag nano composite material is constructed and is used for detecting glucose.

A nanocomposite electrochemical sensor is an electrode loaded with a COOH-G/Au @ Ag material.

In one embodiment, the COOH-G/Au @ Ag material is a nanoparticle in which gold and silver are sequentially coated on the surface of carboxylated graphene.

The preparation method of the nanocomposite electrochemical sensor comprises the following steps:

polishing the surface of the glassy carbon electrode;

and preparing a suspension of the COOH-G/Au @ Ag material, adding the suspension to the surface of the carbon breaking electrode, and drying to obtain the carbon breaking electrode.

In one embodiment, the suspension of the COOH-G/Au @ Ag material has a concentration of 1mg mL^-1。

The application of the nanocomposite electrochemical sensor in detection of a solution containing glucose is disclosed.

In one embodiment, the detection is by electrochemiluminescence.

In one embodiment, the solution contains 1mM glucose.

In one embodiment, the scan rate in the assay is 25-300 nmVs^-1。

In one embodiment, the luminol content of the assay is controlled between 0.1 and 0.6mM, preferably 0.1 to 0.4 mM.

Advantageous effects

A novel COOH-G/Au @ Ag nano material is synthesized by adopting a two-step reduction method. Based on the nano material, a novel enzyme-free ECL sensor which is simple and convenient to operate and low in consumption is prepared. The large catalytic surface area and fast electron transfer rate show very good electrocatalytic oxidation performance for glucose. Furthermore, our sensor not only has good repeatability and a wide linear range, but also has acceptable sensitivity and stability. The prepared sensor for detecting the glucose in the real serum is feasible, and has wide application prospect in the field of clinical diagnosis.

Drawings

FIG. 1 SEM images of (A) (B) (C) COOH-G/Au @ Ag and TEM images of (D) COOH-G/Au @ Ag

FIG. 2 EDX characterization of COOH-G/Au @ Ag

FIG. 3 SEM-EDS mapping image of COOH-G/Au @ Ag nanocomposite

FIG. 4 is a graph of EIS of the detection process. Wherein (A) the I of naked GCE (a, b) with and without glucose and COOH-G/Au @ Ag modified GCE (c, d) with and without glucose_ECLThe curve/E. Illustration is shown: naked GCE (a, b) with no or glucose, and COOH-G/Au @ Ag modified GCE (c, d) with no or glucose CV curves. (B) EIS curves of naked GCE, COOH-G-GCE, COOH-G/Au @ Ag-GCE

FIG. 5 (A) Effect of COOH-G/Au @ Ag amount on chemiluminescence; (B) influence of pH conditions; (C) the effect of luminol concentration on chemiluminescence; (D) the effect of the scan rate.

FIG. 6 interference Capacity of ECL sensor (a-p: glucose, Glycine, ascorbic acid, Urea, Na)⁺，Fe³⁺，Mn²⁺，Cu²⁺，I^-，Zn²⁺，SO₄ ^2-，Pb²⁺Dopamine, alanine, maltose, fructose)

FIG. 7 is a graph of performance during the assay, wherein (A) luminol I on COOH-G/Au @ Ag modified GCE at different glucose concentrations_ECLInsert of the/E curve (a: 0.005. mu.M, b: 0.01. mu.M, c: 0.05. mu.M, d: 0.1. mu.M, E: 0.5. mu.M, f: 1. mu.M, g: 3. mu.M, h: 5. mu.M, i: 8. mu.M, j: 10. mu.M, k: 30. mu.M, l: 50. mu.M, M: 100. mu.M, n: 500. mu.M, o: 800. mu.M, p: 1000. mu.M, q: 1500. mu.M): repeated ECL emissions and continuous CV scans (B) of the linear relationship between glucose concentration (0.005-1500 μ M) were performed on electrodes modified with COOH-G/Au @ Ag composites. Illustration is shown: linear relationship between glucose concentration (0.005-10 μ M).

Detailed Description

Reagent and apparatus

TABLE 1 reagents

TABLE 2 Instrument

Arrangement of the Primary reagents

Preparation of PBS buffer solution

TABLE 3 preparation of PBS buffer solutions at different pH values

Weighing potassium dihydrogen phosphate and sodium hydroxide solids with different pH values and calculated mass by using an analytical balance, adding distilled water for dissolving uniformly, transferring to a 100mL volumetric flask, fixing the volume, and shaking up to prepare PBS buffer solutions with different pH values, wherein the specific preparation method is shown in tables 2-3. After the solution is prepared, label paper is attached, and the solution is placed into a refrigerator for standby.

②5.0mmol L^-1K₃Fe(CN)₆/K₄Fe(CN)₆

0.7456g of KCl, 0.2112g K were weighed out on an analytical balance₄Fe(CN)₆And 0.1646g K₄Fe(CN)₆Putting the crystal into a small beaker, adding a proper amount of distilled water for dissolving, transferring into a 100mL volumetric flask, fixing the volume, shaking up, sticking a label, and putting into a refrigerator for later use.

③0.1mol L^-1NaOH

Weighing 4.000g of NaOH by using an analytical balance, putting the NaOH into a small beaker, dissolving the NaOH by using double distilled water, cooling the solution, transferring the solution into a 100mL volumetric flask, fixing the volume to a scale, shaking the solution uniformly, and putting the solution into a reagent bottle for later use.

④1mmol L^-1C₆H₁₂O₆

0.19817g C6H12O6 is weighed by an analytical balance, placed in a small beaker, dissolved by double distilled water, transferred to a 1L volumetric flask, added to a constant volume to scale, shaken up and placed in a reagent bottle for later use.

⑤1mmol L^-1Luminol

0.0177g of luminol is weighed by an analytical balance, put into a small beaker, dissolved by 0.2M NaOH, then transferred into a 100mL volumetric flask, fixed to the volume to the scale, shaken up, and put into a reagent bottle for later use.

⑥0.1mol·L^-1KCl

0.7455g of KCl is weighed by an analytical balance, put into a small beaker, dissolved by double distilled water, transferred into a 100mL volumetric flask, added to a constant volume to a scale, shaken up and input into a reagent bottle for later use.

Electrode activation

(1) Electrode pretreatment

The pretreatment method comprises the specific steps of uniformly polishing a glassy carbon electrode on sand paper, washing with distilled water, ultrasonically cleaning for 2 minutes, and preparing α -Al with the particle size of 1.0-0.3 mu m₂O₃Dropping appropriate amount of dispersion liquid on the chamois, polishing for 3-5min until the electrode surface is smooth, and treating with 1:1 ethanol and 1:1HNO₃And ultrasonically cleaning in distilled water for 3min until the electrode is cleaned completely.

(2) Electrode activation and detection

After the electrode is cleaned, a three-electrode experimental system is constructed, and 0.5M H is used as an electrolyte solution₂SO₄And (3) setting a potential scanning range of-1.0V-1.0V for the solution, and repeatedly scanning the cyclic voltammetry curve for multiple times until the current response of the curve is stable. Then, the solution was heated to a temperature of 10mmol L^-1K₃Fe(CN)₆And performing cyclic voltammetry scanning in 0.5M KCL solution, determining the difference between the oxidation peak and reduction peak potentials, and determining that the potential difference is less than 80mV, thus the electrode is polished clean.

Preparation of COOH-G @ Au @ Ag nano composite material

Firstly, weighing 6mg of carboxylated graphene (COOH-G) into a clean beaker by using an analytical balance, weighing 50mL of distilled water, pouring the distilled water into the beaker, sealing the beaker by using a preservative film, ultrasonically dispersing the beaker for 6 hours, transferring the uniformly peeled carboxylated graphene suspension into a 100mL three-well flask, setting the oil bath temperature to be 100 ℃, and adding 200 mu L of 2.94 × 10^-2M chloroauric acid (HAuCl)₄) Thereafter, 0.53mL of 3.88 × 10 was added^-2Adding sodium citrate solution to react for 30min, stopping heating, stirring to cool the reaction mixture to room temperature, centrifuging to separate the sample, dissolving in 30mL distilled water, adding 200 μ L1.93 × 10^-2M silver nitrate and 0.3mL of 0.1M Ascorbic Acid (AA) are set to ice bath temperature of 4 ℃, after 2 hours, the mixture is kept stand and stirred for 2 hours at room temperature until the solution becomes dark brown, and after the reaction is finished, the mixture is centrifugally separated, and is dried in vacuum to obtain the required COOH-G/Au @ Ag nano composite material.

Preparation of modified electrode

1mgCOOH-G/Au @ Ag nanocomposite was weighed using an analytical balance to prepare 1mg mL^-1The stock solution of (1) was transferred by a microinjector to 5. mu.L of the COOH-G/Au @ Ag dispersion solution and applied dropwise onto the surface of a glassy carbon electrode, followed by drying at room temperature.

Characterization of COOH-G/Au @ Ag nanomaterial

The morphology and structure of the resulting nanocomposite was studied by SEM and TEM. In FIG. 1, (A), (B) and (C) are scanning electron micrographs at different magnifications. It can be seen that the surface of COOH-G showed a large number of wrinkles, which are spontaneous curling behavior, and the morphology of the wrinkles helps to increase the contact area of glassy carbon. The surface area of the electrode enhances the catalytic performance of the modified electrode. Precious metal Au and Ag nano particles are modified on the surface of COOH-G by two continuous reduction methods to form a compact core-shell structure, and the structure is uniformly adsorbed on the surface of COOH-G with the particle size of about 50 nm. The surface of the trench-like structure functions well as a carrier and adsorbs noble metal nanoparticles more easily, which greatly enhances the electrical conductivity and catalytic performance of the composite. Fig. 1 (D) is a surface morphology feature examined by TEM, which more clearly shows their structure. It indicates that the two elements are loaded on the surface of graphene, and the core-shell structure is confirmed. It can be seen from the transmission electron micrograph that after the reduction reaction, the noble metal Au @ Ag core-shell nanoparticles are regularly adsorbed on the surface of the folds of COOH-G, and the particle size of the formed metal spheres is about 40-50nm, which is consistent with the SEM result. We performed EDX measurements to obtain the chemical composition of the nanomaterials. As shown in fig. 2, the EDX image also shows that the material mainly contains C (62.96%), O (16.11%), Au (13.03%) and Ag (7.91%). SEM-EDS elemental mapping analysis also confirmed that the distribution of Au @ Ag core-shell nanoparticles was high even throughout the analysis area. By performing ICP characterization on the synthesized nanocomposite, the ratio of AuNPs to AgNPs content in the COOH-G/Au @ Ag nanocomposite is about 1: 2, which is similar to the characterization of EDX, further indicating that Au @ Ag core-shell nanoparticles are loaded in COOH-G. SEM, TEM, EDX and ICP images demonstrate that we have successfully synthesized COOH-G/Au @ Ag nanomaterials.

Electrochemical characterization of biosensors

The IECL/E curves of luminol on naked GCE and on COOH-G/Au @ Ag modified GCE prepared without 1mM glucose (with 0.1M, pH 7.4) are shown in panel (A) of FIG. 4. The scan voltage range is-1 to 1V (vs Ag | AgCl | KClsat) and the scan rate is 100mV s^-1. Curve (c) of the COOH-G/Au @ Ag modified GCE without glucose shows that ECL emission is generated by the oxidation product of luminol. In addition, the COOH-G/Au @ Ag modified GCE (curve d) showed a higher spike around 0.35V with 1mM glucose compared to the bare GCE without and with glucose (curves a, b). At the same time, we also investigated its CVs curve (inset of a of fig. 4). As can be seen from the figure, after the glucose is added, obvious oxidation peak current appears in the COOH-G/Au @ Ag modified electrode, which further proves that the COOH-G/Au @ Ag nano material has electrocatalytic oxidation performance on the glucose. This is consistent with the results of ECL experiments.

EIS (electrochemical impedance spectroscopy) is widely used as an effective interface analysis technique for studying electron conductivity between a modified electrode and an electrolyte. Electrode surface resistorChanges in resistance can be obtained by EIS. The interfacial electron transfer resistance of different nanomaterials (GCE, COOH-G/Au, COOH-G/Au @ Ag) on modified GCE was studied by EIS ((B) of FIG. 4). Using a medium containing 5mM K₃Fe(CN)₆/K₄Fe(CN)₆And the AC voltage was set to 10mV (bias potential of 200mV, 10 mV)^-1-10⁵Hz) were subjected to EIS experiments. The diameter of the semicircle in fig. 4(B) reflects the change in the interface electron transfer resistance. The semi-circles in the figure represent the charge transfer process in the high frequency region, which is derived from the ionic resistance of the electrolyte, the interfacial resistance of the modifying material and the interfacial resistance of the modifying electrode and the electrolyte. The straight line represents the diffusion control process in the low frequency region. The smaller the radius of the semicircle in the high frequency region, the stronger the conductivity of the material. As can be seen from fig. 4(B), the impedance of the different modified electrodes in the high frequency part is the electron transfer impedance of the nanomaterial modified electrode, and the impedance value is COOH-G: 305 Ω, COOH-G/Au @ Ag: 218 Ω. Apparently, the impedance value of COOH-G/Au @ Ag is smaller than that of COOH-G, so the electron transport rate at the interface of the GCE modified by COOH-G/Au @ Ag is faster than that of the GCE modified by COOH-G. All these results indicate that the COOH-G/Au @ Ag nanomaterial provides a good electron conduction pathway.

Optimization of experimental conditions

To determine the optimal working environment for a glucose sensor to improve the performance of a glucose sensor assay system, we investigated various factors that may influence the developed sensor response, such as the amount of COOH-G/Au @ Ag, PBS solutions at different pH values, luminol concentration and different scan rates.

(1) Modification gram number of COOH-G/Au @ Ag nano material

To understand the effect of COOH-G/Au @ Ag content in the experiment, we controlled the amount of COOH-G/Au @ Ag to be 0.01-2mg mL^-1Within range, the current response was explored. As shown in FIG. 5 (A), the concentration of the surfactant is 0.01 to 0.05mg mL^-1In the concentration range of (A), the current intensity increases with the modification amount of COOH-G/Au @ Ag, and when the modification amount of COOH-G/Au @ Ag is higher than 0.05mgmL^-1The current response begins to decrease as the concentration increases. One possible cause of this phenomenon is modificationThe concentration of the material is too high to form a thick film on the electrode surface, thereby inhibiting electron transfer. Thus, the optimal material concentration was determined to be 0.05mg mL^-1

(2) Effect of pH of PBS buffer on the System

In fig. 5(B), we investigated the effect of pH on the system. The pH of the buffer solution has a crucial influence on the sensitivity of the sensor. Thus, we prepared PBS buffers at pH 7.0, 7.2, 7.4, 7.6, 7.8 and 8.0 and selected the optimal pH conditions for 1.0mM glucose response on COOH-G/Au @ Ag modified GCE. As shown in fig. 5(B), the ECL intensity will reach a maximum when the pH is 7.4. Based on the experimental results, we finally selected PBS buffer solution with pH 7.4 for the next assay.

(3) Effect of different concentrations of luminol on the System

To determine the luminol concentration that will give the maximum response of the sensor, we controlled the luminol content between 0.1 and 0.6mM using PBS (0.1M, pH 7.4). Fig. 5(C) shows ECL intensity as a function of luminol concentration. As the luminol concentration increased from 0.1mM to 0.4mM, the ECL intensity increased with increasing luminol concentration. However, when the luminol concentration is >0.4mM, the ECL intensity will gradually decrease due to the self-quenching effect. Therefore, a 0.4mM Luminol solution was the best choice throughout the experiment.

(4) Influence of different sweeping speeds on luminous intensity

To optimize the electrocatalytic performance of the sensor, we studied the sweep rate from 25mV s^-1Increased to 300nmVs^-1Effect on the COOH-G/Au @ Ag modified GCE response. As shown in FIG. 5(D), we can see that when the scan rate is at 25mV s^-1To 300mV s^-1Within the range, the ECL strength increases and then decreases. When the scanning rate is 100mV s^-1The ECL intensity showed a maximum. Therefore, 100mV s was chosen in the experiment^-1As the optimum scan rate. From the above experimental results, we can conclude that the optimal conditions for detecting glucose with the GCE modified with COOH-G/Au @ Ag are: 0.05mg mL^-1COOH-G/[email protected]，PBS(0.1M，pH＝7.4)，100mV s^-1And 0.4mM luminol stock solution.

(5) Interference immunity study

When glucose is measured in a sample, substances coexisting in the sample often affect the measurement result to some extent. To verify the selectivity of the ECL sensor, various interfering substances were investigated, including glycine, ascorbic acid, urea, Na⁺，Fe^{3 +}，Mn²⁺，Cu²⁺，I^-，Zn²⁺，SO₄ ^2-，Pb²⁺Dopamine, alanine, maltose and fructose. The results are shown in fig. 6, where the effect of the interfering substances on the detection is negligible and also indicates a better selectivity for glucose.

(6) Detection of glucose by a sensor

As shown in fig. 7 (a), the ECL intensity varies with the glucose concentration under the optimal conditions, and the ECL intensity is found to have a linear relationship with the glucose concentration by linear fitting. The regression equation is that I is 4.27364C +4054.43362 (R)²0.999) ranging from 0.005 μ M to 1.5 mM. As shown in the inset, the linear rule also mixes at lower glucose concentrations. The detection limit (S/N-3) was 0.002. mu.M, and the sensitivity was 15. mu.A. mu.M^-1cm^-2。

At the same time, the reproducibility and stability of ECL sensors were also investigated. As can be seen from fig. 7 (a) (inset), the Relative Standard Deviation (RSD) of the single modified electrode was 0.54% for 7 consecutive measurements of 1M glucose. When 6 independent electrodes were measured, the relative standard deviation was 1.83%, indicating good reproducibility of the sensor. Table 1 lists the linear range, detection limit and sensitivity of COOH-G/Au @ Ag modified GCE compared to most previous reports [1, 10, 18, 31-34 ]. The proposed ECL glucose biosensor has comparable or better analytical performance. Thus, good reproducibility and acceptable durability can be achieved for the developed glucose ECL biosensor.

TABLE 1 comparison of the Linear Range, detection Limit and sensitivity of various glucose

(7) Application of sensor in human serum

In order to finally apply the prepared ECL sensor to the field of detection, we need to assess its applicability and therefore apply it to monitoring glucose levels in human serum samples. Under optimal conditions, we successfully tested the glucose in human serum samples, the results of which are listed in table 2 and compared to the local hospital assay. The recovery rate ranged from 98.36% to 101.23%, indicating that it is expected to be a reliable technique for detecting glucose in human serum samples.

TABLE 2 determination of glucose in human serum samples

The above examples do not represent a limitation of the present invention.