Electrochemiluminescence signal enhancement method based on micellar triple enrichment effect, sensor and application

Abstract

The present application relates to the technical field of electrochemiluminescence sensing, and discloses an electrochemiluminescence signal enhancement method based on micellar triple enrichment effect, a sensor and application, wherein the electrochemiluminescence signal enhancement method based on micellar triple enrichment effect comprises the following steps: placing a working electrode in a solution containing luminol, dissolved oxygen and a hydrophobic target analyte to perform static enrichment, applying a scanning potential to the enriched working electrode, and obtaining an enhanced electrochemiluminescence signal; wherein the working electrode is a working electrode modified by a surfactant micelle-filled vertical ordered mesoporous silica film. The method can simultaneously enrich luminophore, co-reactant and hydrophobic target analyte, significantly enhance the luminol / dissolved oxygen system signal, and has excellent anti-interference ability. The electrochemiluminescence sensor obtained by the method is suitable for efficient and high-sensitivity detection of hydrophobic trace substances in a complex system.

Description

Technical Field

[0001] This invention relates to the field of electrochemiluminescence sensing technology, and in particular to an electrochemiluminescence signal enhancement method, sensor, and application based on the micelle triple enrichment effect. Background Technology

[0002] Morula, a natural flavonoid compound with antioxidant and other biological activities, has significant research value in the prevention and treatment of pregnancy complications, necessitating the development of efficient and sensitive detection methods. Electrochemiluminescence (ECL) analysis is an analytical method that excites a luminescent reaction through electrochemical means. It boasts significant advantages such as low background signal, ease of operation, wide dynamic range, and strong spatiotemporal controllability, making it a commonly used analytical tool in fields such as biosensing, environmental monitoring, and clinical diagnosis. The luminol / dissolved oxygen (DO) system is a common ECL analysis system, where luminol acts as the luminescent agent, and dissolved oxygen participates in the luminescent reaction as an endogenous co-reactant, with both contributing to signal output. However, in practical applications, the luminol / dissolved oxygen system exhibits weak signals and is susceptible to interference from exogenous substances, making it difficult to achieve efficient and sensitive detection of trace substances. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides an electrochemiluminescence signal enhancement method based on the micelle triple enrichment effect, which can significantly enhance the signal of the luminol / dissolved oxygen system and has excellent anti-interference capabilities.

[0004] The first specific technical solution of the present invention is: an electrochemiluminescence signal enhancement method based on the triple enrichment effect of micelles, comprising the following steps: placing the working electrode in a solution containing luminol, dissolved oxygen and hydrophobic target analyte for static enrichment, applying a scanning potential to the enriched working electrode to obtain an enhanced electrochemiluminescence signal; wherein, the working electrode is a working electrode modified with a vertically ordered mesoporous silica film filled with surfactant micelles.

[0005] An electrode modified with a vertically ordered mesoporous silica film filled with surfactant micelles was placed in a mixed solution containing luminol, dissolved oxygen, and a hydrophobic target analyte. The unique composite interface structure of this working electrode enables the simultaneous and selective enrichment of luminol, dissolved oxygen, and the hydrophobic target analyte, achieving triple enrichment of the luminol luminate, the endogenous co-reactant dissolved oxygen, and the hydrophobic target analyte. This significantly enhances the local concentration of the electrochemiluminescence reaction pair in the electrode reaction microregion, resulting in a significant enhancement of the ECL signal. Simultaneously, this working electrode structure exhibits excellent anti-interference capability and selectivity in complex samples, allowing the sensor to generate a stable signal entirely dependent on endogenous dissolved oxygen.

[0006] Optionally, in the vertically ordered mesoporous silica film filled with surfactant micelles, the surfactant micelles are formed from cationic surfactants.

[0007] Optionally, the cationic surfactant is hexadecyltrimethylammonium bromide.

[0008] This surfactant can be used to prepare mesoporous films with pore sizes of 2-3 nm, which have high specific surface areas and can achieve the filling of a large number of micelles.

[0009] Optionally, the working electrode is prepared by electrochemically assisted self-assembly, that is, a vertically ordered mesoporous silica film filled with surfactant micelles is grown on a conductive substrate by electrochemically assisted self-assembly (EASA).

[0010] The EASA method can obtain highly ordered, vertically interconnected nanochannels, while the mesoporous structures grown by Stöber solution have slightly lower order than those grown by the EASA method.

[0011] Optionally, the preparation time of the electrochemical-assisted self-assembly method is 5 to 25 seconds, that is, the growth time of the vertically ordered mesoporous silica film filled with surfactant micelles on the conductive substrate is 5 to 25 seconds, preferably 5 to 10 seconds.

[0012] Optionally, the conductive substrate of the working electrode is indium tin oxide (ITO).

[0013] ITO is low in cost and has a stable electrochemical window and high light transmittance.

[0014] Optionally, the settling enrichment time is 90~120s.

[0015] When the settling enrichment time is less than 90s, the enrichment is incomplete, the electrode background ECL signal is low, and the detection linear range is reduced; when the settling enrichment time is more than 120s, the detection time is increased and the detection efficiency is reduced.

[0016] Optionally, the concentration of luminol in the solution containing luminol, dissolved oxygen, and the hydrophobic target analyte is 100-150 μM.

[0017] The concentration of luminol affects the ECL signal of the electrode. When the concentration is below 100 μM, the background ECL signal of the electrode is low, which reduces the detection linear range; while when the concentration is above 150 μM, it increases the detection cost.

[0018] Optionally, the range of the scanning potential is -1.0 to +0.8 V.

[0019] Furthermore, the scanning direction is 0 V → -1 V → +0.8 V.

[0020] The second specific technical solution of the present invention is: an electrochemiluminescence sensor, comprising a working electrode modified with a vertically ordered mesoporous silica film filled with surfactant micelles, wherein the working electrode performs the above-mentioned electrochemiluminescence signal enhancement method based on the micelle triple enrichment effect.

[0021] This electrochemiluminescence sensor can be used for the efficient and highly sensitive detection of trace hydrophobic substances.

[0022] The third specific technical solution of the present invention is: the application of an electrochemiluminescence sensor in the detection of morin for non-diagnostic purposes, wherein the electrochemiluminescence sensor is the aforementioned electrochemiluminescence sensor, and includes the following steps: the electrochemiluminescence sensor is the aforementioned electrochemiluminescence sensor, and includes the following steps: placing the working electrode of the electrochemiluminescence sensor in a solution containing luminol, dissolved oxygen and morin for static enrichment, applying a scanning potential to the working electrode, exciting and recording the electrochemiluminescence signal, and determining the morin content based on the change of the electrochemiluminescence signal relative to the background signal; wherein the background signal is the electrochemiluminescence baseline signal measured in a detection system containing only luminol and dissolved oxygen.

[0023] Morin is a hydrophobic target analyte. This invention utilizes the enrichment effect of the working electrode on morin, causing it to aggregate at high concentrations in the electrode micro-regions. The enriched morin molecules effectively quench the electrochemiluminescence (ECL) signal generated by the luminol / dissolved oxygen system through electron transfer or energy transfer mechanisms, resulting in a significant reduction in ECL intensity. By monitoring the degree of signal quenching relative to the background baseline, highly sensitive and selective quantitative detection of morin can be achieved. This detection method not only greatly improves the detection sensitivity and lowers the detection limit of morin by leveraging the triple enrichment effect of the triple enrichment working electrode, but also effectively eliminates the influence of other interfering substances in complex matrices through the selective recognition capability of the electrode interface, ensuring the accuracy and reliability of the detection results.

[0024] Compared with the prior art, the present invention has at least the following advantages: (1) In the prior art, vertically ordered mesoporous silica films are usually used for traditional electrochemical detection and as enrichment carriers for single components. This invention innovatively modifies electrodes with surfactant micelle-filled vertically ordered mesoporous silica films and applies them to the field of ECL signal enhancement. Based on this structure, a complete technical solution suitable for ECL detection is constructed. For the first time, triple enrichment of luminol luminescent material, dissolved oxygen endogenous co-reactant, and hydrophobic target analyte is achieved, which significantly improves the local concentration of electrochemiluminescence reaction pairs in the electrode reaction micro-region, promotes significant enhancement of ECL signal, and shows excellent anti-interference ability and selectivity in complex samples. It overcomes the inherent defects of weak signal and poor anti-interference ability of luminol / dissolved oxygen system, and enables it to be used for efficient and sensitive detection of hydrophobic trace substances in complex systems. It has important scientific value and practical application prospects. (2) In order to obtain the best detection performance, the present invention systematically optimized the growth time of the vertically ordered mesoporous silica film filled with surfactant micelles on the conductive substrate, the enrichment time of the working electrode and the concentration of luminol, etc., to achieve efficient enrichment of luminol, dissolved oxygen and hydrophobic target analytes, and to achieve a significant reduction in the detection limit. The experimental results show that, taking the model analyte morin as the object of investigation, the detection limit of this method is as low as 0.18 pg / mL, and the linear range covers 6 orders of magnitude (1 pg / mL to 1 μg / mL). (3) The composite structure of the working electrode is robust, the micelles are not easily lost, and the ECL signal remains highly stable during continuous scanning; (4) The electrochemiluminescence sensor of the present invention does not require activation or complex modification and can be directly used for rapid detection of target substances in complex samples, such as urine. Attached Figure Description

[0025] Figure 1 These are morphological images of the SM@VMSF / ITO and SM@SNF / ITO electrodes; Figure 2 These are the ATR-FTIR spectra of ITO, SM / ITO, VMSF / ITO, and SM@VMSF / ITO electrodes; Figure 3 These are contact angle diagrams for ITO, SM@VMSF / ITO, and VMSF / ITO electrodes; Figure 4 The electrodes are ITO, VMSF / ITO, and SM@VMSF / ITO in the presence of [Fe(CN)6]. 3- CV signal graph in 0.1 M KCl solution; Figure 5 The electrodes are ITO, VMSF / ITO, and SM@VMSF / ITO in the presence of [Ru(NH3)6]. 3+CV signal graph in 0.1 M KCl solution; Figure 6 The image shows the CV signal diagrams of ITO, VMSF / ITO, and SM@VMSF / ITO electrodes in a 0.1 M KCl solution containing hydroxymethyl ferrocene. Figure 7 This is an ECL signal diagram of ITO, VMSF / ITO, and SM@VMSF / ITO electrodes in PBS buffer solution containing luminol; Figure 8 These are CV signal graphs of ITO, VMSF / ITO, and SM@VMSF / ITO electrodes after immersion in different solutions; Figure 9 These are ECL signal images of ITO, VMSF / ITO, and SM@VMSF / ITO electrodes after immersion in different solutions; Figure 10 These are CV signal graphs of SM@VMSF / ITO under different gas atmospheres; Figure 11 These are ECL signal graphs of SM@VMSF / ITO under different gas atmospheres; Figure 12 This is a graph showing the relationship between ECL signal and potential under different scanning directions; Figure 13 This is an ECL response diagram of SM@VMSF / ITO with different free radical scavengers added to 0.01 M PBS buffer solution; Figure 14 This is a comparison chart of the stability of SM@VMSF / ITO, SM / ITO, and VMSF / ITO electrodes; Figure 15 This is a graph of the ECL signal of SM@VMSF / ITO after BSA processing at different times; Figure 16 This is a bar chart comparing the ECL signals of SM@VMSF / ITO and VMSF / ITO after adding 1 μg / mL morin to PBS test solution containing luminol; Figure 17 This is an ECL signal diagram of SM@VMSF / ITO electrodes grown at different times; Figure 18 It is a graph of ECL signals generated by enriching luminol at different times; Figure 19 This is a comparison of ECL signals of ITO, VMSF / ITO, and SM@VMSF / ITO electrodes under different concentrations of luminol; Figure 20 This is an ECL signal graph of SM@VMSF / ITO in morin solutions of different concentrations; Figure 21 This is a line graph showing the relationship between different morin concentrations corresponding to SM@VMSF / ITO and VMSF / ITO and ECL signals. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0027] Unless otherwise specified, all raw materials used in the following specific embodiments were purchased commercially and used directly without special processing. The main raw materials used in the embodiments are as follows: Tetraethyl orthosilicate (TEOS, 99%), hexadecyltrimethylammonium bromide (CTAB), potassium ferricyanide (K3[Fe(CN)6]), potassium ferrocyanide (K4[Fe(CN)6]), hexaammineruthenium ([Ru(NH3)6]Cl3), sodium dihydrogen phosphate hydrate (NaH2PO4·2H2O), disodium hydrogen phosphate hydrate (Na2HPO4·12H2O), ammonia (NH3·H2O, 28%), anhydrous sodium acetate (NaAc), glacial acetic acid (HAc, 99%), sodium hydroxide (NaOH), luminol, morin (Mor, 95%), isopropanol (IPA), p-benzoquinone (BQ), bovine serum albumin (BSA), sodium chloride (NaCl), glucose (Glu), uric acid (UA), ascorbic acid (AA), and starch (Starch) were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). Acetone (Me₂CO₃), anhydrous ethanol (99.8%), and concentrated hydrochloric acid (HCl, 38%) were purchased from Shuanglin Chemical Reagent Co., Ltd. (Hangzhou, China). Sodium chloride (NaCl), sodium nitrate (NaNO₃), and potassium chloride (KCl) were purchased from Gaojing Fine Chemical Co., Ltd. (Hangzhou, China). Phosphate-buffered saline (PBS) was prepared from Na₂HPO₄ and NaH₂PO₄. All chemicals and reagents were of analytical grade and were not subjected to additional treatment before use. ITO glass electrodes were purchased from Zhuhai Kaiwei Optoelectronic Technology Co., Ltd. (Guangdong, China).

[0028] Example 1: The present invention provides an electrochemiluminescence signal enhancement method based on the micelle triple enrichment effect, comprising the following steps: placing the working electrode in a solution containing luminol, dissolved oxygen and hydrophobic target analyte for static enrichment, applying a scanning potential to the enriched working electrode to obtain an enhanced electrochemiluminescence signal; wherein, the working electrode is a working electrode modified with a vertically ordered mesoporous silica film filled with surfactant micelles.

[0029] The preparation process of the above-mentioned working electrode is as follows: First, the ITO glass electrode is pretreated to obtain the supporting electrode. The ITO glass electrode is then immersed in 1 M NaOH solution overnight, followed by ultrasonic cleaning in acetone, anhydrous ethanol, and ultrapure water for 10 min each. After drying, it is cut to the target size using a glass cutter. Mesoporous channel films are prepared using the EASA method and the Stöber solution growth method, respectively. For distinction, the vertically ordered mesoporous silica film prepared by the EASA method is abbreviated as VMSF, and the vertically ordered mesoporous silica film prepared by the Stöber solution growth method is abbreviated as SNF. The surfactant micelles are written as SM. Based on the above materials, SM@VMSF / ITO electrodes and SM@SNF / ITO electrodes are prepared, respectively.

[0030] Specifically, the SM@VMSF / ITO electrode was prepared using an electrochemically assisted self-assembly method. 20 mL of ethanol was mixed with 20 mL of 0.1 M NaNO3 aqueous solution, followed by the addition of 1.585 g CTAB and 3.05 mL TEOS. The mixture was stirred vigorously at room temperature for 2.5 h to obtain the precursor solution. The three-electrode system was completely immersed in the prepared precursor solution, with the working electrode being an indium tin oxide (ITO) electrode. This ITO electrode had dimensions of 0.5 cm × 5 cm, and its effective working area was defined as 0.5 cm × 1 cm using insulating tape. An application of -0.7 mA·cm⁻¹ was applied to the working electrode. -2 A constant current was applied for 10 seconds to initially modify the electrode surface. The constant current application time can be 5–25 seconds. The modified electrode was then removed from the precursor solution and thoroughly rinsed with ultrapure water to remove adsorbed unreacted precursors and impurities. The cleaned electrode was then aged at 120°C for 12 hours. After these steps, the nanochannels of the resulting electrode were loaded with surfactant micelles (SM). This modified electrode is designated SM@VMSF / ITO.

[0031] Specifically, the SM@SNF / ITO electrode was prepared using the Stöber solution growth method. 160 mg of CTAB was weighed and added to an ethanol-water mixture (30 mL anhydrous ethanol and 70 mL water), and stirred until the CTAB was completely dissolved. Then, 80 μL of TEOS was added to the solution, and stirring was continued for 5 min to obtain the precursor solution. A 2.5 cm × 5 cm ITO electrode was completely immersed in the precursor solution, sealed, and placed at 60 °C for 24 h to complete the electrode surface modification. After the reaction, the electrode was removed from the precursor solution and thoroughly rinsed with ultrapure water to remove adsorbed unreacted reagents and impurities. The cleaned electrode was then aged at 100 °C for 12 h to obtain the SM@SNF / ITO electrode. The SM@SNF / ITO electrode was cut into 0.5 × 5 cm pieces; insulating tape was used to define the working area as 0.5 × 1 cm.

[0032] The SM@VMSF / ITO or SM@SNF / ITO working electrodes were placed in a solution containing luminol, dissolved oxygen, and a hydrophobic target analyte for static enrichment. The luminol concentration was 100 μM, and the enrichment time was controlled at 100 s. The CTAB micelles were used to simultaneously enrich luminol, dissolved oxygen, and the hydrophobic target analyte at the electrode interface, achieving triple enrichment and increasing the local concentration of the reactants. In other embodiments, the luminol concentration in the solution containing luminol, dissolved oxygen, and the hydrophobic target analyte was 100–150 μM, and the static enrichment time was 90–120 s.

[0033] Cyclic voltammetry scan potentials of -1.0 to +0.8 V (vs. Ag / AgCl) were applied to the enriched working electrode at a scan rate of 100 mV / s to excite electrochemiluminescence and record the signal simultaneously. A significantly enhanced ECL signal was obtained through the triple enrichment effect. Furthermore, the scan was performed in the direction of 0 V → -1 V → +0.8 V to ensure the synergistic reaction between reactive oxygen species and luminol radicals and improve the signal enhancement effect.

[0034] Example 2: This invention provides an electrochemiluminescence sensor, comprising a counter electrode, a reference electrode, and a working electrode modified with a vertically ordered mesoporous silica film filled with surfactant micelles. The working electrode performs the electrochemiluminescence signal enhancement method based on the micelle triple enrichment effect as described in Example 1. In this example, the working electrode is the SM@VMSF / ITO electrode described in Example 1; in another example, the working electrode is the SM@SNF / ITO electrode described in Example 1.

[0035] Example 3: This invention provides the application of an electrochemiluminescence sensor in the detection of morin for non-diagnostic purposes. The electrochemiluminescence sensor is the same as that described in Example 2, and includes the following steps: placing the SM@VMSF / ITO electrode of the electrochemiluminescence sensor in a solution containing luminol, dissolved oxygen, and morin for static enrichment; applying a scanning potential to the working electrode to excite and record the electrochemiluminescence signal; and determining the morin content based on the change of the electrochemiluminescence signal relative to the background signal; wherein the background signal is the electrochemiluminescence baseline signal measured in a detection system containing only luminol and dissolved oxygen.

[0036] The background signal detection process involves adding luminol to 0.01 M, pH 7.4 phosphate buffer (PBS) and adjusting the concentration to 100 μM to form a base solution containing natural dissolved oxygen. The SM@VMSF / ITO electrode of the sensor is immersed in the base solution, and cyclic voltammetry (CV) is used to excite ECL and simultaneously measure ECL at a potential range of -1.0 ~ +0.8 V (vs. Ag / AgCl). The scan rate is 100 mV / s, and the scan direction is 0 V → -1 V → +0.8 V to obtain the background signal.

[0037] In this embodiment, urine samples from healthy adults were taken, and a known concentration of morin standard solution was added. The solution was then diluted 100 times with 0.01 M, pH 7.4 phosphate buffer to prepare the test sample solution. A known concentration of morin standard solution was added to the test sample solution to prepare a spiked sample solution. The SM@VMSF / ITO working electrode of the electrochemiluminescence sensor was placed sequentially in the test sample solution, and static enrichment and potential scanning were performed under the same conditions as the background signal. The corresponding ECL signals were recorded. Based on the changes in ECL signals before and after spiking, combined with the added morin standard concentration, the actual morin content in the urine sample was calculated. Multiple parallel spiked detections (n=3) were performed, and the spiked recovery rate and relative standard deviation (RSD) were calculated to verify the accuracy and precision of the detection method. In this invention, the recovery rate was between 103% and 105%, and the RSD was between 0.5% and 3.8%, indicating that the detection results were accurate and reproducible.

[0038] To verify the effectiveness of the electrochemiluminescence signal enhancement method of the present invention, a series of tests were conducted. In the following tests, the SM@VMSF / ITO electrode was the SM@VMSF / ITO electrode prepared by the EASA method in Example 1; the SM@SNF / ITO was the SM@SNF / ITO prepared by the Stöber solution growth method in Example 1; SM / ITO was CTAB-modified ITO without nanochannels; VMSF / ITO was an ITO electrode modified with a vertically ordered mesoporous silica film without surfactant micelles; and ITO was a bare indium tin oxide glass electrode without any surface modification layer.

[0039] The morphology of the SM@VMSF / ITO and SM@SNF / ITO electrodes prepared in Example 1 was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the results are as follows: Figure 1 As shown in the figure, (A) is a SEM image of the SM@VMSF / ITO electrode interface, clearly showing the three-layer sandwich structure of the glass layer, ITO layer, and SM@VMSF layer. The thickness of the SM@VMSF layer is approximately 92 nm. (B) and (C) are top-view TEM images of the SM@VMSF layer, showing that its film has a uniform hexagonal ordered mesoporous structure with a pore diameter of approximately 2-3 nm. The surface is intact and crack-free, and the film thickness is consistent with the SEM characterization results. (D) and (E) are TEM images of the SM@SNF layer. Unlike the SM@VMSF layer, the SM@SNF layer does not exhibit a long-range hexagonal ordered structure. Its pores are arranged in a disordered worm-like pattern, but the pore diameter is also approximately 2-3 nm. The film is also continuous and crack-free, with a thickness of approximately 113 nm. The characterization results indicate that the film prepared by the EASA method has a highly ordered vertical mesoporous structure, while the Stöber method forms disordered worm-like pores. Given that highly ordered channels are beneficial for the orderly filling of micelles and the uniform and rapid transport of reactants, the present invention prefers the EASA method as the preparation method for SM@VMSF electrodes.

[0040] ITO, VMSF / ITO, SM@VMSF / ITO, and SM / ITO were analyzed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The results are as follows: Figure 2 As shown, Si-O-Si peaks appeared in both VMSF / ITO and SM@VMSF / ITO, confirming the silicon dioxide structure. SM@VMSF / ITO showed a peak at 2920 cm⁻¹. -1 and 2850 cm -1 The C–H stretching vibration peak consistent with that of pure CTAB appeared at the point, while the signal disappeared in the VMSF / ITO film with micelles removed, proving that the CTAB micelles were successfully confined inside the mesoporous channels.

[0041] Contact angle tests were performed on ITO, VMSF / ITO, and SM@VMSF / ITO, and the results are as follows: Figure 3 As shown, the contact angle of SM@VMSF / ITO is 75°, which is higher than that of ITO (58°) and VMSF / ITO (43°), indicating a significant enhancement in its surface hydrophobicity. This improved property is mainly attributed to the partially exposed hydrophobic alkyl chains in the micelles.

[0042] The electrochemical properties of ITO, SM@VMSF / ITO, and VMSF / ITO electrodes in different probe solutions were investigated using cyclic voltammetry. The results are as follows: Figure 4 , Figure 5 and Figure 6 As shown in the figure, bare ITO exhibits obvious reversible redox peaks in all three probe solutions. For SM@VMSF / ITO, no Faradaic current was observed for either the anion probe K3[Fe(CN)6] or the cation probe [Ru(NH3)6]Cl3. The neutral hydrophobic probe hydroxymethyl ferrocene exhibits obvious redox peaks on the SM@VMSF / ITO electrode. The VMSF / ITO electrode shows redox peaks for the probe in all three probe solutions. This proves that SM@VMSF / ITO, formed after filling micelles, selectively permeates and enriches only neutral hydrophobic molecules through the hydrophobic enrichment effect of the micelles.

[0043] The ECL signal intensity of ITO, SM@VMSF / ITO, and VMSF / ITO electrodes in luminol-containing PBS buffer solution was investigated, and the results are as follows: Figure 7 As shown, the ECL signal measured by SM@VMSF / ITO is significantly enhanced compared to other electrodes, indicating that the introduction of micelles has a significant sensitizing effect on the ECL response.

[0044] The enrichment capacity of SM@VMSF / ITO for luminol was tested using cyclic voltammetry and electrochemiluminescence, and the results are as follows: Figure 8 and Figure 9 As shown, Figure 8 When the three electrodes were soaked in PBS buffer without luminol, no obvious redox peaks were observed. Only SM@VMSF / ITO showed a significant oxidation peak near +0.8 V after being soaked in PBS buffer containing luminol, which proves that the micelles in the SM@VMSF / ITO structure have excellent enrichment ability for luminol. Figure 9 The results further validated the above inference.

[0045] The ECL signal of SM@VMSF / ITO was measured by cyclic voltammetry and electrochemiluminescence in saturated luminol-PBS solutions under N2, air, and O2 atmospheres, respectively. The results are as follows: Figure 10 and Figure 11 As shown, it can be seen that the ECL signal is significantly enhanced with the increase of O2 concentration in the solution, which proves that dissolved oxygen, as a co-reactant, synergistically enhances the ECL signal with luminol.

[0046] The relationship between ECL signal intensity and potential scan direction was tested, and the results are as follows: Figure 12 As shown, the electrode obtained the highest ECL response signal when the potential was scanned from 0 V to -1 V and then back to +0.8 V. If the scanning direction was reversed, the signal decreased significantly. This is because near -1 V, dissolved oxygen undergoes an oxygen reduction reaction to generate reactive oxygen species (ROS). These ROS have a relatively long lifetime in aqueous solution and can accumulate on and near the electrode surface. Subsequently, when the potential is scanned back to +0.8 V, luminol is oxidized to generate its anionic free radical. This intermediate has a short lifetime and decays rapidly. Therefore, when the scan first passes through a negative potential and then enters a positive potential, the pre-accumulated ROS and the newly generated luminol free radicals undergo a chemiluminescent reaction, thus obtaining a strong ECL signal.

[0047] The SM@VMSF / ITO electrode was placed in an ECL test system, and 100 μM concentrations of either benzoquinone (BQ) or isopropanol (IPA) were added to the solution. BQ specifically captures superoxide radicals, while IPA effectively quenches hydroxyl radicals. Results are as follows: Figure 13 As shown, the ECL signal decreased significantly after the addition of BQ, while the signal did not change significantly after the addition of IPA. When both BQ and IPA were added simultaneously, the ECL signal also decreased significantly, indicating that the key active species in this ECL system is mainly the superoxide anion radical. Figure 13 I and I0 are the ECL signals with or without free radical scavengers, respectively; PMT = 700 V, and the scan rate is 100 mV / s.

[0048] During continuous potential scanning, the ECL signals of the three electrodes, SM@VMSF / ITO, SM / ITO, and VMSF / ITO, changed as follows: Figure 14 As shown. Compared with SM / ITO and VMSF / ITO electrodes, the ECL signal of the SM@VMSF / ITO electrode is not only highly stable with a relative standard deviation (RSD) of only 0.4%, but also has a significantly improved signal strength, proving that the composite structure of the working electrode of this invention is robust and the micelles are not easily lost.

[0049] To investigate the anti-interference ability of the SM@VMSF / ITO electrode in complex biological matrices, 50 mg / mL bovine serum albumin (BSA) was selected as a model protein contaminant. ECL signals were measured after incubating the SM@VMSF / ITO electrode with BSA solution for different times. The results are as follows: Figure 15As shown, the ECL signal is basically consistent with the initial value, indicating that the electrode can effectively resist protein contamination. This is mainly attributed to the intact, crack-free structure of the VMSF film and the blocking effect of SM micelles on the nanochannels. The two work together to prevent the penetration and adsorption of macromolecules on the electrode surface, thereby significantly reducing matrix interference.

[0050] To verify the quenching effect of morin on the ECL signal and the enrichment effect of the SM@VMSF / ITO electrode on morin, morin was added to luminol solution, and the ECL responses of the SM@VMSF / ITO and VMSF / ITO electrodes were compared. The results are as follows: Figure 16 As shown, the inset is a magnified histogram of the ECL signal for VMSF / ITO. From Figure 16 As can be seen, after adding 1 μg / mL morin, the ECL signal of SM@VMSF / ITO decreased to 18.5% of the initial signal, while that of VMSF / ITO only decreased to 55.3%. The results indicate that morin can effectively quench the ECL signal of the luminol / dissolved oxygen system, and the quenching efficiency of SM@VMSF / ITO is significantly higher. This is attributed to the enrichment effect of SM micelles on morin, which can significantly increase its local concentration on the electrode surface, thereby enhancing the quenching effect and laying the foundation for high-sensitivity detection of morin.

[0051] This invention also tested the effects of surfactant micelle-filled vertically ordered mesoporous silica films (SM@VMSF) on detection performance under varying conditions, including growth time on a conductive substrate, enrichment time at the working electrode, and luminol concentration. The results are as follows: Figures 17-19 As shown. From Figure 17 It can be seen that the ECL signal is significantly enhanced when the growth time is 5-10 seconds; from Figure 18 It can be seen that the ECL signal tends to stabilize when the enrichment time of the working electrode is 90~120s. Figure 19 It can be seen that when the concentration of luminol is 100~150μM, the ECL signal intensity is significantly improved and gradually tends to stabilize. Further increasing the concentration has no significant effect on signal enhancement.

[0052] The electrochemiluminescence sensor prepared in Example 2 was used to detect ECL in mulberry pigment solutions of different concentrations. The results are shown in Figure 20: The ECL signal gradually decreased with increasing mulberry pigment concentration. Within the range of 1 pg / mL to 1 μg / mL, the ECL signal showed a good linear relationship with the mulberry pigment concentration. Based on the corresponding linear regression equation and a signal-to-noise ratio (S / N) of 3, the detection limit for mulberry pigment by this sensor was 0.18 pg / mL. As a control, parallel detection was performed under the same conditions using an electrochemiluminescence sensor with a VMSF / ITO working electrode. Figure 21It is known that the linear range of the VMSF / ITO electrode for morin is 10 pg / mL to 100 ng / mL, and its detection limit is calculated to be 1.5 pg / mL according to the corresponding linear regression equation. Comparing the detection performance of the two electrodes, it can be seen that the electrochemiluminescence sensor of this invention effectively broadens the linear detection range and significantly improves the detection sensitivity.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for enhancing electrochemiluminescence signals based on the micelle triple enrichment effect, characterized in that, Includes the following steps: The working electrode was placed in a solution containing luminol, dissolved oxygen, and a hydrophobic target analyte, namely morin, for static enrichment. A scanning potential was applied to the enriched working electrode to obtain an enhanced electrochemiluminescence signal. The working electrode was modified with a vertically ordered mesoporous silica film filled with surfactant micelles. In the vertically ordered mesoporous silica film filled with surfactant micelles, the surfactant micelles are formed by a cationic surfactant; the cationic surfactant is hexadecyltrimethylammonium bromide. The working electrode is prepared by electrochemical-assisted self-assembly, and the surfactant micelles are loaded in the nanochannels of the working electrode; the static enrichment time is 90~120s.

2. The electrochemiluminescence signal enhancement method based on the micelle triple enrichment effect according to claim 1, characterized in that, The preparation time for the electrochemical-assisted self-assembly method is 5 to 25 seconds.

3. The electrochemiluminescence signal enhancement method based on the micelle triple enrichment effect according to claim 1, characterized in that, The concentration of luminol in the solution containing luminol, dissolved oxygen, and the hydrophobic target analyte is 100-150 μM.

4. The electrochemiluminescence signal enhancement method based on the micelle triple enrichment effect according to any one of claims 1 to 3, characterized in that, The range of the scanning potential is -1.0 ~ +0.8 V.

5. An electrochemiluminescence sensor, characterized in that, The invention includes a working electrode modified with a vertically ordered mesoporous silica film filled with surfactant micelles, and the working electrode performs the electrochemiluminescence signal enhancement method based on the micelle triple enrichment effect as described in any one of claims 1 to 4.

6. The application of an electrochemiluminescence sensor in the detection of morin for non-diagnostic purposes, characterized in that, The electrochemiluminescence sensor is the electrochemiluminescence sensor according to claim 5, comprising the following steps: placing the working electrode of the electrochemiluminescence sensor in a solution containing luminol, dissolved oxygen and morin for static enrichment; applying a scanning potential to the working electrode to excite and record the electrochemiluminescence signal; and determining the content of morin based on the change of the electrochemiluminescence signal relative to the background signal; wherein the background signal is the electrochemiluminescence baseline signal measured in a detection system containing only luminol and dissolved oxygen.

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