Molecularly imprinted electrochemical luminescence sensor as well as preparation method and application thereof

Abstract

The invention discloses a molecularly imprinted electrochemical luminescence sensor as well as a preparation method and application thereof, and belongs to the technical field of electrochemical luminescence. According to the invention, terpyridyl ruthenium doped silicon nanoparticles Ru (at) SiO2 are used as a luminescent material, tripropylamine is used as a co-reaction reagent, and a domoic acid molecularly imprinted polymer MIP is used as a recognition element. The preparation method comprises the following steps: sequentially coating Ru-coated SiO2 and MIP on a glassy carbon electrode (GCE) to obtain an MIP / Ru-coated SiO2 / GCE sensing interface; and finally, incubating in a solution containing domoic acid to obtain the DA / MIP / Ru coated SiO2 / GCE electrochemical luminescence biosensor which is used as a working electrode for electrochemical luminescence test. The method has a wide detection linear range, high sensitivity and good reproducibility, can realize trace analysis of domoic acid, and provides an innovative and sensitive analysis platform for detection of domoic acid in an actual sample.

Description

Technical Field

[0001] The present invention belongs to the field of electrochemiluminescence analysis and detection, and in particular relates to a molecular imprinting electrochemiluminescence sensor and a preparation method and application thereof. Background Art

[0002] Domoic acid (C 15 H 21 Domoic acid (NO6) is a natural amino acid excitatory neurotoxin produced primarily by the diatom Pseudo-rhomboid diatom and is the primary component of amnesic shellfish toxins. Consuming shellfish contaminated with domoic acid can cause adverse health effects in humans, including dizziness, vomiting, memory loss, epilepsy, and even death, posing a serious threat to human health and food safety. Currently, the main detection methods for domoic acid include MBA, ELISA, HPLC, LC-MS, fluorescence, and electrochemistry. However, these methods often suffer from complex operation, high detection costs, low sensitivity, and severe background interference, making them unsuitable for trace analysis of biomolecules in actual samples. Therefore, there is an urgent need to develop a simple, low-cost, and highly sensitive detection method.

[0003] Electrochemiluminescence (ECL) technology is a detection method that combines the principles of electrochemistry and chemiluminescence. By applying a voltage, a luminescent reagent on the electrode surface undergoes electron transfer, forming excited species that emit light radiation, thereby enabling qualitative and quantitative analysis of the target. Its core advantages include high sensitivity, low background noise, strong controllability, fast response, and a wide linear range. It is ideally suited for the quantitative detection of trace amounts of target substances and exhibits broad application potential in food safety and public health. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a molecular imprinting electrochemiluminescence sensor that can detect domoic acid quickly, simply, sensitively and with high specificity.

[0005] The technical problem that the present invention also aims to solve is to provide a method for preparing the molecular imprinting electrochemiluminescence sensor.

[0006] The final technical problem to be solved by the present invention is to provide an application of the molecular imprinting electrochemiluminescence sensor in detecting the content of domoic acid in a sample.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a molecular imprinting electrochemiluminescence sensor, which includes MIP / Ru@SiO2 / GCE as a working electrode, a platinum wire electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode. The MIP / Ru@SiO2 / GCE is obtained by modifying Ru@SiO2 / GCE with MIP, and the Ru@SiO2 / GCE is obtained by modifying a glassy carbon electrode with Ru@SiO2.

[0008] The amount of the MIP added is 5 to 10 μL, and the concentration of the MIP is 0.5 to 3 mg / L.

[0009] The diameter of the glassy carbon electrode is 3 to 4 mm, the Ru@SiO2 / GCE is obtained by modifying the Ru@SiO2 luminescent material onto GCE, the concentration of the Ru@SiO2 solution is 0.5 to 3 mg / mL, and the drop coating amount of the Ru@SiO2 luminescent material is 5 to 10 μL.

[0010] The present invention also includes a method for preparing the molecularly imprinted electrochemiluminescence sensor, comprising the following steps:

[0011] (1) Weigh Ru@SiO2 and disperse it ultrasonically in ultrapure water, then drop-coat it on the surface of a glassy carbon electrode and let it dry to obtain Ru@SiO2 / GCE.

[0012] (2) MIP was weighed and ultrasonically dispersed in chitosan-acetic acid aqueous solution, and then drop-coated onto the surface of Ru@SiO2 / GCE and dried to obtain MIP / Ru@SiO2 / GCE.

[0013] The preparation process of the Ru@SiO2 material in step (1) is as follows: Ru(bpy)3Cl2 solution, ultrapure water, ammonia water and anhydrous ethanol are mixed and stirred for 10-60 minutes, and then TEOS is added and stirred in the dark for 6-18 hours to obtain the product Ru@SiO2.

[0014] The concentration of the Ru(bpy)3Cl2 solution is 0.01-1M, the dosage is 0.5 mL, the ultrapure water is 1-5 mL, the ammonia water is 2-8 mL, the anhydrous ethanol is 50-200 mL, the TEOS is 2-8 mL, and the reaction temperature is 25°C.

[0015] Wherein, step (1) further comprises centrifuging the product Ru@SiO2, washing and drying it with ethanol and ultrapure water, and storing it at 4°C in the dark.

[0016] The preparation process of the MIP material is as follows: adding the functional monomer 4-VP to the template molecule 1,3,5-pentanetricarboxylic acid solution (PTA), ultrasonically mixing, and standing at room temperature for 0.5 to 4 hours to form a prepolymer; then, adding water, Span80 and Tween80 to a round-bottom flask respectively, stirring for 5 to 60 minutes to form an emulsion; adding the prepolymer and the crosslinker EGDMA to the emulsion in sequence while stirring, and deoxygenating with nitrogen for 5 to 30 minutes, heating the water bath, and adding the initiator AIBN when the water temperature rises to 60°C. The reaction is stirred in a constant temperature water bath for 12 to 24 hours, adding methanol to the reaction system to break the emulsion, collecting the precipitate by centrifugation, and extracting the obtained polymer with a methanol-acetic acid mixture for 12 to 36 hours to elute the template molecule, and then extracting the polymer with methanol for 6 to 24 hours until it is neutral to obtain the MIP.

[0017] The molar ratio of the 1,3,5-pentanetricarboxylic acid solution to the functional monomer 4-VP is 1:1 to 2:1.

[0018] Wherein, the concentration of the MIP solution in step (2) is 0.5-3 mg / mL, and the drop-coating amount of the MIP solution is 5-10 μL.

[0019] The concentration of the chitosan-acetic acid aqueous solution in step (2) is 0.2%. The preparation process of the 0.2% chitosan-acetic acid aqueous solution material is as follows: 0.5 mL of glacial acetic acid is added to a 50 mL empty volumetric flask, and the volume is adjusted to 50 mL with ultrapure water to obtain a 1% glacial acetic acid solution. 0.1 g of chitosan powder is weighed and added to a 50 mL empty volumetric flask, 30 mL of 1% glacial acetic acid solution is added, and ultrasonically dissolved. After the dissolution is completed, the volume is adjusted to 50 mL with 1% glacial acetic acid solution to obtain a 0.2% chitosan-acetic acid aqueous solution.

[0020] The present invention also includes the application of the molecular imprinting electrochemiluminescence sensor in the detection of domoic acid content.

[0021] The MIP / Ru@SiO2 / GCE working electrode is immersed in a standard solution of domoic acid of different concentrations prepared with ultrapure water for an incubation time of 10 to 60 minutes, and the concentration of the domoic acid is 10 ng / mL to 5000 ng / mL.

[0022] The present invention also includes a method for detecting domoic acid using the molecularly imprinted electrochemiluminescence sensor, comprising the following steps:

[0023] (1) The unincubated MIP / Ru@SiO2 / GCE and the MIP / Ru@SiO2 / GCE incubated with domoic acid were placed in the detection base solution in sequence, and the standard working curve was drawn according to the change value of the electrochemiluminescence signal;

[0024] (2) The content of domoic acid in the pre-treated sample was detected using the drawn standard working curve.

[0025] Specifically, step (1) comprises the following steps: forming a three-electrode system with a working electrode, a reference electrode and an auxiliary electrode in a molecularly imprinted electrochemiluminescence sensor; using a 10×PBS buffer solution (containing 20 mmol / L tripropylamine TPA) with a pH of 7.4 as a detection base solution; placing unincubated MIP / Ru@SiO2 / GCE and MIP / Ru@SiO2 / GCE incubated with domoic acid molecules in the detection base solution in sequence; performing cyclic scanning in a voltage range of 0 to 1.4 V at a scan rate of 100 mV / s and a photomultiplier tube of 400 V, and recording the corresponding ECL signal; and drawing a ΔI-lgC working curve with the logarithm lgC of the concentration of the domoic acid standard solution as the horizontal axis and the change value ΔI of the electrochemiluminescence signal as the vertical axis.

[0026] Specifically, step (2) is to detect the content of domoic acid in the pre-treated sample using the ΔI-1gC working curve drawn in step (1).

[0027] Wherein, the analytes include seawater samples and / or shellfish samples.

[0028] The seawater sample pretreatment method comprises the following steps: filtering the seawater sample using a 0.22 μm aqueous microporous filter membrane, and collecting the filtrate for determination.

[0029] The pretreatment method of the shellfish sample includes the following steps: weighing 10g (accurate to 0.01g) of dried shellfish meat, adding 10mL of 1×PBS (pH7.4), high-speed homogenization for 3min, vortex oscillation for 2min, centrifugation at 6000rpm for 10min, removing the supernatant, filtering with a 0.22μm aqueous microporous filter membrane, and collecting the filtrate for determination.

[0030] Mechanism of the present invention: Ru(bpy)3 2+ The luminescent material exhibits high luminescence efficiency and strong signal intensity, a wide potential window compatibility, and wide environmental adaptability, making it suitable for applications in complex biological systems. The use of silica spheres to coat ruthenium terpyridine molecules significantly enhances their stability, improves biocompatibility, and reduces aggregation of ruthenium terpyridine nanoparticles, thereby enhancing their electrochemiluminescence performance. Using pseudo-template technology, the structural analog of domoic acid (PTA) was used as a template molecule instead of the expensive and highly toxic domoic acid, significantly reducing preparation costs and improving safety.

[0031] The detection principle of the present invention is as follows: First, the Ru@SiO2 luminescent material is used to modify GCE to produce Ru@SiO2 / GCE. Then, MIP is used to modify Ru@SiO2 / GCE to produce MIP / Ru@SiO2 / GCE. MIP modification to the Ru@SiO2 / GCE surface creates an imprinted cavity before incubation of domoic acid molecules. The co-reactant, TPA, reacts with the Ru@SiO2 through the imprinted cavity, emitting an electrochemiluminescent signal. After incubation of the sensor with domoic acid molecules, the molecules rebind to the electrode surface through the specific molecularly imprinted cavity, blocking electron transfer channels and reducing the ECL signal, thereby enabling specific quantitative detection of domoic acid.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention adopts MIP / Ru@SiO2 / GCE as the working electrode to construct a molecular imprinting electrochemiluminescence sensor that can detect domoic acid quickly, simply, sensitively and with high specificity. When the molecular imprinting electrochemiluminescence sensor of the present invention is used for actual sample detection, it shows good specificity and stability, and the minimum detection limit for domoic acid is 3.08 ng / mL. It is suitable for rapid and accurate detection of domoic acid in a variety of samples. The molecular imprinting electrochemiluminescence sensor prepared by the present invention has both high sensitivity and rapid response characteristics. The integrated design significantly simplifies the pre-treatment steps and operation procedures. While achieving low cost, it can efficiently screen domoic acid in a variety of samples, providing a new solution for on-site rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the preparation principle of the domoic acid molecular imprinted electrochemiluminescence sensor of the present invention.

[0034] Figure 2 This is a graph showing the effect of the molar ratio of template molecule PTA and functional monomer 4-VP on the electrochemiluminescence intensity.

[0035] Figure 3 Figure 3 shows the effect of the concentration of domoic acid molecularly imprinted polymer on the electrochemiluminescence intensity.

[0036] Figure 4 ECL response curve of MIP / Ru@SiO2 / GCE to different concentrations of domoic acid (A) and its working curve (af: 10, 100, 500, 1000, 5000, 10000 ng / mL) (B).

[0037] Figure 5 This is the effect of the incubation time of MIP / Ru@SiO2 / GCE in domoic acid standard solution on the electrochemiluminescence intensity.

[0038] Figure 6For electrodes with different treatments, 1mM [Fe(CN)6] 3 / 4- Electrochemical impedance spectroscopy (EIS) in the probe solution (A) and ECL spectra in 10× PBS (pH 7.4) containing 20 mmol / L TPA (ae: bare GCE, Ru@SiO2 / GCE, electrode without PTA washout, MIP / Ru@SiO2 / GCE, and electrode after re-incubation with domoic acid) (B).

[0039] Figure 7 Figure 2 shows the change in the electrochemiluminescence signal ΔI of MIP / Ru@SiO2 / GCE in the presence of 500 ng / mL domoic acid (DA) and 5000 ng / mL saxitoxin (STX), okadaic acid (OA), and tetrodotoxin (TTX) (A); the electrochemiluminescence response of 6 different electrodes (B); the ECL value of the prepared electrode tested after incubation with 500 ng / mL domoic acid every two days after being stored for two weeks (C); the ECL graph of the sensor scanned continuously for 8 cycles after incubation with 100 ng / mL and 5000 ng / mL domoic acid, respectively (D). DETAILED DESCRIPTION

[0040] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0041] Example 1 Preparation of MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor

[0042] (1) 0.5 mL of 0.01 M Ru(bpy)3Cl2 solution, 3 mL of ultrapure water, 4.8 mL of ammonia, and 150 mL of anhydrous ethanol were mixed and stirred for 20 min. 4.5 mL of TEOS was then added and stirred at 25°C in the dark for 12 h to obtain the product Ru@SiO2. Finally, the product (Ru@SiO2) was centrifuged and washed with ethanol and ultrapure water to obtain Ru@SiO2 material, which was stored at 4°C in the dark.

[0043] (2) Weigh 54.045 mg of the template molecule PTA (1,3,5-pentanetricarboxylic acid, Shanghai MacLean Biochemical Technology Co., Ltd., Product No. P865357) and dissolve it in 2 mL of methanol / acetonitrile (1:1, V:V) organic solvent. Add 107.8 μL of the functional monomer 4-VP (4-vinylpyridine, Shanghai MacLean Biochemical Technology Co., Ltd., Product No. V820414), mix by ultrasonication for 20 min, and let stand at room temperature for 2 h to form a prepolymer. Then, add 25 mL of water, 100 μL of Span80 (Shanghai Aladdin Biochemical Technology Co., Ltd., Product No. S110839), and 100 μL of Tween80 (Shanghai MacLean Biochemical Technology Co., Ltd., Product No. T6336) to a 150 mL round-bottom flask, respectively, and stir for 30 min to form an emulsion. To the above emulsion, add the prepolymer and 1414.51 μL of crosslinker EGDMA (ethylene glycol dimethacrylate, Shanghai MacLean Biochemical Technology Co., Ltd., Product No. E808858) in sequence, stirring while adding. After nitrogen deoxygenation for 15 minutes, heat the water bath. When the water temperature reaches 60°C, add initiator AIBN (2,2'-azobisisobutyronitrile, Shanghai MacLean Biochemical Technology Co., Ltd., Product No. A800354).

[0044] 0.08 g. Maintain a constant temperature of 60°C in a water bath with stirring (600 rpm) for 18 hours. Add 25 mL of methanol to the reaction system to break the emulsion. Centrifuge (12,000 rpm, 10 minutes) to collect the precipitate. The resulting polymer is Soxhlet extracted with a mixture of methanol and acetic acid (9:1, V:V) for 24 hours to elute the template molecules. The polymer is then extracted with methanol for 12 hours until neutral to obtain a molecularly imprinted polymer (MIP). Dry in a 60°C oven for 12 hours, then pass through a 200-mesh sieve. The sieved MIP powder is collected for later use.

[0045] (3) Add 0.5 mL of glacial acetic acid to a 50 mL empty volumetric flask and dilute to 50 mL with ultrapure water to obtain a 1% glacial acetic acid solution. Weigh 0.1 g of chitosan powder (Shanghai MacLean Biochemical Technology Co., Ltd., Cat. No. C804726) and add it to a 50 mL empty volumetric flask. Add 30 mL of 1% glacial acetic acid solution and sonicate to dissolve. After dissolution is complete, dilute to 50 mL with 1% glacial acetic acid solution to obtain a 0.2% chitosan-acetic acid aqueous solution.

[0046] (4) A glassy carbon electrode (GCE) was polished with 200 nm alumina powder and then ultrasonically treated with anhydrous ethanol and deionized water. After being blown dry, 8 μL of Ru@SiO2 solution (prepared with 1.0 mg of Ru@SiO2 material and ultrapure water to prepare a 1.0 mg / mL Ru@SiO2 solution) was drop-coated and dried at room temperature to obtain Ru@SiO2 / GCE. Next, 8 μL of MIP solution (prepared with 2 mg of MIP material and a 0.2% chitosan-acetic acid aqueous solution to prepare a 2 mg / mL MIP solution) was drop-coated and dried at room temperature to obtain a MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor, which served as the working electrode for electrochemiluminescence testing.

[0047] Example 2 Preparation of MIP / Ru@SiO2 / GCE Molecularly Imprinted Electrochemiluminescence Sensor

[0048] The amount of template molecule PTA added in step (2) of Example 1 was adjusted to 25.5225 mg, and other conditions remained unchanged to obtain MIP powder.

[0049] Example 3 Preparation of MIP / Ru@SiO2 / GCE Molecularly Imprinted Electrochemiluminescence Sensor for Detection of Domoic Acid

[0050] The amount of template molecule PTA added in step (2) of Example 1 was adjusted to 102.09 mg, and other conditions remained unchanged to obtain MIP powder.

[0051] Example 4 Preparation of MIP / Ru@SiO2 / GCE Molecularly Imprinted Electrochemiluminescence Sensor

[0052] The amount of template molecule PTA added in step (2) of Example 1 was adjusted to 204.18 mg, and other conditions remained unchanged to obtain MIP powder.

[0053] Example 5 Preparation of MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor

[0054] The amount of template molecule PTA added in step (2) of Example 1 was adjusted to 408.36 mg, and other conditions remained unchanged to obtain MIP powder.

[0055] In order to improve the selectivity of the ECL sensor, the present invention uses MIP as an element that specifically recognizes domoic acid. When MIP is modified on the electrode surface, it will hinder the contact between the luminescent material Ru@SiO2 and the co-reactant TPA, thereby reducing the intensity of electrochemiluminescence. After the PTA molecules are eluted from the MIP, the exposed imprinted holes will increase the contact area between the luminescent material Ru@SiO2 and the co-reactant TPA, enhance the efficiency of luminescence, and ultimately increase the intensity of electrochemiluminescence. When the domoic acid molecules in the object to be detected are re-bound to the MIP, the intensity of electrochemiluminescence is reduced again. Figure 2 As shown in the figure, when the molar ratio of PTA to 4-VP is 1:2, the luminescence intensity change ΔI after adsorption of the same concentration of domoic acid is the largest, so the molar ratio of PTA to 4-VP of 1:2 is selected as the optimal ratio parameter.

[0056] Example 6 Preparation of MIP / Ru@SiO2 / GCE Molecularly Imprinted Electrochemiluminescence Sensor

[0057] (1) 0.5 mL of 0.1 M Ru(bpy)3Cl2 solution, 1 mL of ultrapure water, 1.6 mL of ammonia, and 50 mL of anhydrous ethanol were mixed and stirred for 10 min. 1.5 mL of TEOS was then added and stirred at 25°C in the dark for 12 h. Finally, the product (Ru@SiO2) was centrifuged and washed with ethanol and ultrapure water to obtain Ru@SiO2 material, which was then stored at 4°C in the dark.

[0058] (2) Weigh 108.09 mg of the template molecule PTA and dissolve it in 2 mL of methanol / acetonitrile (1:1, V:V) organic solvent. Add 107.8 μL of the functional monomer 4-VP, mix thoroughly by ultrasonication for 20 min, and let it stand at room temperature for 4 h to form a prepolymer. Then, add 25 mL of water, 100 μL of Span80, and 100 μL of Tween80 to a 150 mL round-bottom flask, respectively, and stir for 30 min to form an emulsion. Add the prepolymer solution and 1414.51 μL of the crosslinker EGDMA to the above emulsion in sequence, stirring while adding. After nitrogen deoxygenation for 30 min, heat the water bath. When the water temperature rises to 60°C, add 0.08 g of the initiator AIBN. Maintain a constant temperature of 60°C in a water bath with stirring (600 rpm) and react for 12 h. Add 25 mL of methanol to the reaction system to break the emulsion. Centrifuge (12,000 rpm, 10 minutes) to collect the precipitate. Soxhlet extraction of the resulting polymer with a mixture of methanol and acetic acid (9:1, V:V) for 18 hours to elute the template molecule. Extract the polymer with methanol for 24 hours until neutral to obtain the MIP. Dry the mixture in a 60°C oven for 24 hours, then pass it through a 200-mesh sieve. The sieved MIP powder is collected for later use.

[0059] (3) Add 0.5 mL of glacial acetic acid to a 50 mL empty volumetric flask and dilute to 50 mL with ultrapure water to obtain a 1% glacial acetic acid solution. Weigh 0.1 g of chitosan powder and add it to a 50 mL empty volumetric flask. Add 30 mL of 1% glacial acetic acid solution and dissolve it by ultrasonication. After dissolution is complete, dilute to 50 mL with 1% glacial acetic acid solution to obtain a 0.2% chitosan-acetic acid aqueous solution.

[0060] (4) The GCE was polished with 200 nm alumina powder and then ultrasonically treated with anhydrous ethanol and deionized water. After being blown dry, 8 μL of Ru@SiO2 solution (prepared by weighing 1.0 mg of Ru@SiO2 material and ultrapure water to prepare a 1.0 mg / mL Ru@SiO2 solution) was drop-coated and dried at room temperature to obtain Ru@SiO2 / GCE. Next, 8 μL of MIP solution (prepared by weighing 2 mg of MIP material and 0.2% chitosan-acetic acid aqueous solution to prepare a 2 mg / mL MIP solution) was drop-coated and dried at room temperature to obtain a MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor, which served as the working electrode for electrochemiluminescence testing.

[0061] Example 7 Preparation of MIP / Ru@SiO2 / GCE Molecularly Imprinted Electrochemiluminescence Sensor

[0062] The concentration of MIP prepared in step (4) of Example 6 was adjusted to 1 mg / mL, while other conditions remained unchanged to obtain a MIP solution.

[0063] Example 8 Preparation of MIP / Ru@SiO2 / GCE Molecularly Imprinted Electrochemiluminescence Sensor

[0064] The concentration of MIP prepared in step (4) of Example 6 was adjusted to 1.5 mg / mL, while other conditions remained unchanged, to obtain a MIP solution.

[0065] Example 9 Preparation of MIP / Ru@SiO2 / GCE Molecularly Imprinted Electrochemiluminescence Sensor

[0066] The concentration of MIP prepared in step (4) of Example 6 was adjusted to 2.5 mg / mL, while other conditions remained unchanged, to obtain a MIP solution.

[0067] Example 10 Preparation of MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor

[0068] The concentration of MIP prepared in step (4) of Example 6 was adjusted to 3 mg / mL, while other conditions remained unchanged to obtain a MIP solution.

[0069] Through the above examples 6 to 10, it was found that when the amount of the MIP solution added was 8 μL, the optimal concentration was 2 mg / mL. When the amount added was small, MIP would not be able to effectively block the reaction between Ru@SiO2 and the coreactant TPA, and the change in ΔI before and after adsorption was low. However, when the amount added was large, MIP would excessively block the reaction between Ru@SiO2 and the coreactant TPA, and the change in ΔI before and after adsorption was low. Figure 3 As shown in the figure, the best effect was achieved when the drop volume was 8 μL and the concentration was 2 mg / mL.

[0070] Example 11 Application of MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor

[0071] (1) detecting the response intensity of the molecularly imprinted electrochemiluminescence sensor in solutions containing different concentrations of domoic acid, wherein the concentration of domoic acid in the target solution ranges from 10 ng / mL to 5000 ng / mL;

[0072] (2) Drawing of standard curve

[0073] The Ag / AgCl electrode and the platinum wire electrode were used as the reference electrode and the counter electrode, respectively, and the MIP / Ru@SiO2 / GCE prepared in Example 1 was used as the working electrode to form a three-electrode system. A 10×PBS buffer solution (containing 20 mmol / LTPA) with a pH of 7.4 was used as the detection substrate. The MIP / Ru@SiO2 / GCE working electrode was immersed in a standard solution of different concentrations of domoic acid prepared with ultrapure water and incubated for 30 minutes. The unincubated MIP / Ru@SiO2 / GCE and the MIP / Ru@SiO2 / GCE incubated with domoic acid molecules were placed in the detection substrate successively, and cyclic scanning was performed in the voltage range of 0 to 1.4 V at a scan rate of 100 mV / s and a photomultiplier tube of 400 V, and the corresponding ECL signal change value ΔI was recorded.

[0074] The ΔI-lgC working curve was plotted, with the logarithm of the domoic acid concentration, log C, as the horizontal axis and the change in the electrochemiluminescence signal, ΔI, as the vertical axis. The sensor's lower detection limit (LOD) was calculated to be 3.08 ng / mL. The domoic acid content in the sample corresponding to the change in the electrochemiluminescence signal was calculated using the following formula: ΔI = 1535.42 log C + 2378.48. See the graph for the curve. Figure 4 .

[0075] (3) Actual sample testing

[0076] 3.1. The seawater samples were filtered using a 0.22 μm aqueous microporous membrane and the filtrate was collected for determination.

[0077] 3.2. First, separate the scallop tissue sample. Weigh 10 g (accurate to 0.01 g) of sample and add 10 mL of 1×PBS (pH 7.4). Homogenize at high speed for 3 minutes, vortex for 2 minutes, and centrifuge at 6000 rpm for 10 minutes. Remove the supernatant and filter through a 0.22 μm aqueous microporous filter membrane. Collect the filtrate for determination. Replace the domoic acid standard solution with the sample solution and determine the domoic acid content in the sample solution using the ΔI-1gC working curve drawn in step (2).

[0078] Example 12 Application of MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor

[0079] The incubation time in the standard solution of domoic acid in step (2) of Example 11 was adjusted to 10 min, and the other conditions remained unchanged, and the test was carried out.

[0080] Example 13 Application of MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor

[0081] The incubation time in the standard solution of domoic acid in step (2) of Example 11 was adjusted to 20 min, and the other conditions remained unchanged, and the test was carried out.

[0082] Example 14 Application of MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor

[0083] The incubation time in the standard solution of domoic acid in step (2) of Example 11 was adjusted to 45 min, and the other conditions remained unchanged, and the test was carried out.

[0084] Example 15 Application of MIP / Ru@SiO2 / GCE molecularly imprinted electrochemiluminescence sensor

[0085] The incubation time in the standard solution of domoic acid in step (2) of Example 11 was adjusted to 60 min, and the other conditions remained unchanged, and the test was carried out.

[0086] The electrode incubation time in Examples 11-15 is 1 to 60 minutes, with 30 minutes being the optimal time. Figure 5 As shown in the figure, when the incubation time is less than 30 minutes, ΔI increases with the incubation time, reaching its maximum value at 30 minutes and remaining almost unchanged after 30 minutes. This indicates that the imprinted cavity reaches saturation after 30 minutes, so 30 minutes was selected as the optimal incubation time.

[0087] Example 16

[0088] Electrochemical impedance spectroscopy and ECL tests were used to characterize the bare GCE (a), Ru@SiO2 / GCE (b), electrode without PTA washout (c), MIP / Ru@SiO2 / GCE (d), and MIP / Ru@SiO2 / GCE electrode re-incubated with 1.0 μg / mL domoic acid (e).

[0089] The experiment set up a control group and 4 treatment groups:

[0090] Control group (a): bare GCE electrode was used as blank control.

[0091] Treatment 1(b): 8 μL of the dispersed Ru@SiO2 luminescent material solution prepared in Example 1 with a concentration of 1 mg / mL was dropwise applied to the surface of the bare glassy carbon electrode and allowed to dry to obtain a modified Ru@SiO2 / GCE electrode;

[0092] Treatment 2(c): 2 mg of MIP material was weighed and a 2 mg / mL MIP solution was prepared with a 0.2% chitosan-acetic acid aqueous solution. 8 μL of the dispersed 0.2% chitosan-acetic acid aqueous solution was drop-coated on the surface of a bare glassy carbon electrode and allowed to dry to obtain a modified MIP / Ru@SiO2 / GCE electrode.

[0093] Treatment 3 (d): 8 μL of the 2 mg / mL MIP solution prepared in Example 1 and dispersed in chitosan-acetic acid aqueous solution was drop-coated on the surface of the Ru@SiO2 / GCE electrode and allowed to dry to obtain a modified MIP / Ru@SiO2 / GCE electrode.

[0094] Treatment 4(e): The eluted MIP / Ru@SiO2 / GCE working electrode prepared in Example 1 was immersed in a standard solution of domoic acid and incubated for 30 min, and then dried to obtain an electrode re-incubated with domoic acid.

[0095] The results of the experiments were respectively carried out on bare GCE, Ru@SiO2 / GCE, MIP / Ru@SiO2 / GCE, electrodes without PTA washing and electrodes after re-incubation with domoic acid at 1 mM [Fe(CN)6] 3 / 4- Electrochemical impedance spectroscopy (ECL) spectra of the probe in solution and in 10×PBS (pH 7.4) containing 20 mmol / L TPA.

[0096] Depend on Figure 6As can be seen in Figure A, compared to bare GCE, the impedance of Ru@SiO2 / GCE is higher, indicating that Ru@SiO2 has been successfully modified on the electrode surface. The electrode with uneluted PTA has the highest impedance, indicating that the introduction of the molecularly imprinted membrane hinders electron transfer. The impedance of MIP / Ru@SiO2 / GCE is lower, indicating that the exposed molecularly imprinted cavity can serve as a channel for electron transfer, accelerating the electron transfer rate. Upon rebinding with domoic acid, the molecularly imprinted cavity is blocked, hindering electron transfer and increasing the impedance. This demonstrates the successful fabrication of the molecularly imprinted electrochemiluminescence sensor.

[0097] Depend on Figure 6 As shown in Figure B, compared to bare GCE, Ru@SiO2 / GCE exhibits the strongest ECL signal. However, after modification with the molecularly imprinted polymer (MIP) without eluting PTA, the ECL signal significantly decreases. The ECL signal of MIP / Ru@SiO2 / GCE recovers significantly, indicating that the exposed MIP cavity serves as a channel for electron transfer. Upon rebinding with domoic acid, the MIP cavity becomes blocked, hindering electron transfer and causing the ECL signal to decrease again. This demonstrates the successful fabrication of the molecularly imprinted electrochemiluminescence sensor.

[0098] Example 17

[0099] 1. Selective testing

[0100] The MIP / Ru@SiO2 / GCE prepared in Example 1 was incubated with 500 ng / mL domoic acid and 5000 ng / mL other marine toxins (STX, OA, TTX) and then subjected to ECL testing. The test results are as follows: Figure 7 As shown in A.

[0101] 2. Repeatability test

[0102] According to the preparation method of Example 1, 6 MIP / Ru@SiO2 / GCE were prepared under the same conditions for ECL testing. The test results are shown in Figure 7 B.

[0103] 3. Stability test

[0104] (1) The MIP / Ru@SiO2 / GCE prepared according to the preparation method of Example 1 was stored at room temperature for two weeks. After incubation with 500 ng / mL domoic acid every two days, ECL testing was performed for fourteen consecutive days. The test results are shown in Table 1. Figure 7 C.

[0105] (2) According to the preparation method of Example 1, the prepared MIP / Ru@SiO2 / GCE was incubated in 100 ng / mL and 5000 ng / mL of domoic acid, respectively, and scanned continuously for 8 cycles. The test results are shown in FIG. Figure 7 D.

[0106] 4. Results Analysis

[0107] Depend on Figure 7 As shown in Figure 4, the quenching value of the sensor to domoic acid is the largest, and there is no obvious response to other common marine toxins. This proves that the molecularly imprinted electrochemiluminescence sensor has good selectivity for domoic acid.

[0108] Depend on Figure 7 B shows that the ECL responses of six identical MIP / Ru@SiO2 / GCEs were measured under the same conditions, and the relative standard deviation (RSD) of the ECL intensity of the six sensors was less than 2.83%, proving that the sensor has good repeatability.

[0109] Depend on Figure 7 C shows that after the prepared sensor was stored at room temperature for two weeks, the sensor can still maintain 98% of the original ECL response, indicating that the sensor has good storage stability.

[0110] Depend on Figure 7 D shows that the ECL intensity of the sensor does not change much after 8 consecutive cycles of scanning, indicating that the sensor has good stability.

[0111] Application Example 1

[0112] In order to verify the feasibility of the molecular imprinting electrochemiluminescence sensor MIP / Ru@SiO2 / GCE of the present invention in actual sample detection, the domoic acid content of seawater samples was tested.

[0113] The seawater sample was tested using the standard addition method. The seawater sample was filtered using a 0.22 μm aqueous microporous membrane. The filtrate was collected and then added to the domoic acid standard solution for determination. A three-electrode system was used during the determination process. The molecular imprinting electrochemiluminescence sensor MIP / Ru@SiO2 / GCE prepared in Example 1 was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the platinum wire electrode was used as the counter electrode. The ECL test was performed by immersing the MIP / Ru@SiO2 / GCE in the prepared seawater sample for incubation. 10×PBS (pH 7.4) containing 20 mmol / L TPA was used as the detection base solution. The unincubated MIP / Ru@SiO2 / GCE and the MIP / Ru@SiO2 / GCE incubated with domoic acid molecules were placed in the detection solution in sequence. The voltage range was 0-1.4 V, the scan rate was 100 mV / s, and the photomultiplier tube was 400 V. The corresponding electrochemiluminescence signal change value ΔI was recorded. Substitute it into the working curve: ΔI=1535.42lgC+2378.48( Figure 4 ), the content of domoic acid in the seawater sample was calculated. The test results are shown in Table 1.

[0114] Table 1 Detection of domoic acid in seawater samples

[0115] sample DA concentration (ng / mL) DA test results (ng / mL) Recovery rate (%) RSD (%) 1 100 98.06 98.06 7.10 2 500 506.49 101.30 7.49 3 5000 4931.77 98.64 5.15

[0116] The test results show that the molecular imprinting electrochemiluminescence sensor MIP / Ru@SiO2 / GCE of the present invention can detect trace amounts of domoic acid in seawater, which can provide technical support for marine environmental monitoring agencies and fill the gap in this field.

[0117] Application Example 2

[0118] In order to verify the feasibility of the molecular imprinting electrochemiluminescence sensor MIP / Ru@SiO2 / GCE of the present invention in actual sample detection, the domoic acid content of mussel samples was tested.

[0119] The shellfish samples were tested using the standard addition method: 10 g (accurate to 0.01 g) of dried shellfish meat was weighed and added to 10 mL of 1×PBS (pH 7.4). The mixture was homogenized at high speed for 3 min, vortexed for 2 min, and centrifuged at 6000 rpm for 10 min. The supernatant was removed and filtered through a 0.22 μm aqueous microporous filter membrane. The filtrate was collected for determination. During the determination process, a three-electrode system was used. The MIP / Ru@SiO2 / GCE prepared in Example 1 was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the platinum wire electrode was used as the counter electrode. The ECL test was performed by immersing the MIP / Ru@SiO2 / GCE in the prepared seawater sample for incubation. 10×PBS (pH 7.4) containing 20 mmol / L TPA was used as the detection base solution. The unincubated MIP / Ru@SiO2 / GCE and the MIP / Ru@SiO2 / GCE incubated with domoic acid molecules were placed in the detection base solution in sequence. The voltage range was 0-1.4 V, the scan rate was 100 mV / s, and the photomultiplier tube was 400 V. The corresponding electrochemiluminescence signal change value ΔI was recorded. It was inserted into the working curve: ΔI = 1535.42logC + 2378.48 ( Figure 4 ), and the content of domoic acid in the sample was calculated. The test results are shown in Table 2.

[0120] Table 2 Detection of domoic acid in mussel samples

[0121] sample DA concentration (ng / mL) DA test results (ng / mL) Recovery rate (%) RSD (%) 1 100 97.59 97.59 7.76 2 500 518.74 103.75 8.24 3 5000 5029.81 100.60 5.59

[0122] The test results show that the molecular imprinting electrochemiluminescence sensor MIP / Ru@SiO2 / GCE of the present invention can detect trace amounts of domoic acid in mussel seafood, which can provide technical support for food safety regulatory departments and fill the gap in this field.

Claims

1. A molecularly imprinted electrochemiluminescence sensor, characterized in that: The molecular imprinting electrochemiluminescence sensor includes MIP / Ru@SiO2 / GCE as a working electrode, a platinum wire electrode as a counter electrode, and an Ag / AgCl electrode as a reference electrode. The MIP / Ru@SiO2 / GCE is obtained by modifying Ru@SiO2 / GCE with MIP, and the Ru@SiO2 / GCE is obtained by modifying a glassy carbon electrode with Ru@SiO2.

2. The molecularly imprinted electrochemiluminescence sensor according to claim 1, characterized in that The amount of the MIP added is 5 to 10 μL, and the concentration of the MIP is 0.5 to 3 mg / L.

3. The molecularly imprinted electrochemiluminescence sensor according to claim 1, characterized in that The diameter of the glassy carbon electrode is 3-4 mm. The Ru@SiO2 / GCE is obtained by modifying the Ru@SiO2 luminescent material onto GCE. The concentration of the Ru@SiO2 solution is 0.5-3 mg / mL, and the drop coating amount of the Ru@SiO2 luminescent material is 5-10 μL.

4. The method for preparing a molecularly imprinted electrochemiluminescence sensor according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Weigh Ru@SiO2 and disperse it ultrasonically in ultrapure water, then drop-coat it on the surface of a glassy carbon electrode and let it dry to obtain Ru@SiO2 / GCE. (2) MIP was weighed and ultrasonically dispersed in chitosan-acetic acid aqueous solution, and then drop-coated onto the surface of Ru@SiO2 / GCE and dried to obtain MIP / Ru@SiO2 / GCE.

5. The method for preparing a molecularly imprinted electrochemiluminescence sensor according to claim 4, wherein: The preparation process of the Ru@SiO2 material described in step (1) is: mixing Ru(bpy)3Cl2 solution, ultrapure water, ammonia water and anhydrous ethanol and stirring for 10-60 minutes, then adding TEOS and stirring in the dark for 6-18 hours to obtain the product Ru@SiO2.

6. The method for preparing a molecularly imprinted electrochemiluminescence sensor according to claim 4, wherein: The preparation process of the MIP material is as follows: adding the functional monomer 4-VP to a solution of the template molecule 1,3,5-pentanetricarboxylic acid, ultrasonically mixing, and standing at room temperature for 0.5-4 hours to form a prepolymer; then, adding water, Span80, and Tween80 to a round-bottom flask respectively, stirring for 5-60 minutes to form an emulsion; adding the prepolymer and the crosslinker EGDMA to the emulsion in sequence while stirring, and deoxygenating with nitrogen for 5-30 minutes, heating the water bath, and adding the initiator AIBN when the water temperature reaches 60°C. The reaction is stirred in a constant temperature water bath for 12-24 hours, adding methanol to the reaction system to break the emulsion, collecting the precipitate by centrifugation, and extracting the obtained polymer with a methanol-acetic acid mixture for 12-36 hours to elute the template molecule. The polymer is then extracted with methanol for 6-24 hours until it is neutral to obtain the MIP.

7. The method for preparing a molecularly imprinted electrochemiluminescence sensor according to claim 6, wherein: The molar ratio of the 1,3,5-pentanetricarboxylic acid solution to the functional monomer 4-VP is 1:1 to 2:

1.

8. The method for preparing a molecularly imprinted electrochemiluminescence sensor according to claim 4, wherein: The concentration of the MIP solution in step (2) is 0.5-3 mg / mL, and the drop-coating amount of the MIP solution is 5-10 μL.

9. Use of the molecularly imprinted electrochemiluminescence sensor according to any one of claims 1 to 3 in detecting domoic acid content.

10. The use according to claim 9, characterized in that The MIP / Ru@SiO2 / GCE working electrode is immersed in a standard solution of domoic acid of different concentrations prepared with ultrapure water for an incubation time of 10 to 60 minutes, and the concentration of the domoic acid is 10 ng / mL to 5000 ng / mL.

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