Closed split magnetic bipolar electrochemical luminescence sensor and application thereof in detection of kaempferol in safflower carthamus

Abstract

The invention belongs to the technical field of bipolar electrochemical luminescence sensors, and particularly relates to a closed split magnetic bipolar electrochemical luminescence sensor and application thereof in detection of kaempferol in safflower. According to the bipolar electrochemical luminescence sensor, under the action of driving voltage, kaempferol is subjected to electrochemical oxidation reaction on the surface of the anode of the detection chamber to promote oxygen reduction reaction at the cathode end of the bipolar electrode, so that electrons enriched on the electrode surface of the anode end of the bipolar electrode are increased, and generated Faraday current flows through the bipolar electrode; the electrochemical luminescence behavior of Ru (bpy) 32 + / TPrA (at) Au NPs is triggered in the signal chamber, and the electrochemical luminescence intensity is in direct proportion to the concentration of the kaempferol, so that the detection of the kaempferol is realized. The sensor constructed by the invention has the advantages of low cost, wide linear range, low detection limit, high sensitivity, good stability, good reproducibility and the like, and is a novel kaempferol detection method with extremely high application potential.

Description

Technical Field

[0001] This invention belongs to the technical field of bipolar electrochemiluminescence sensors, specifically relating to a closed, split-type magnetically attracted bipolar electrochemiluminescence sensor and its application in the detection of kaempferol in safflower. Background Technology

[0002] Electrochemiluminescence (ECL) refers to the light emission phenomenon that occurs when precursor substances on an electrode surface generate corresponding high-energy excited-state molecules under the excitation of an electrochemical reaction, or when products react with each other after the reaction, or when substances in solution react with the generated products to generate excited-state molecules, which then return to the ground state. As the name suggests, its reaction process consists of two stages: an electrochemical reaction and a chemiluminescence reaction. The electrochemical reaction provides reaction intermediates, while the chemiluminescence reaction generates these intermediates into excited-state substances, thereby exciting the light emission phenomenon. ECL detection methods combine electrochemical and chemiluminescence sensing technologies, thus inheriting the advantages of electrochemical analysis methods (high controllability and fast response) and chemiluminescence methods (high sensitivity, good selectivity, and simple equipment construction). However, traditional three-electrode systems have certain limitations, such as high cost, complex instruments, and cross-reaction interference between the target analyte and the ECL luminescent material / co-reactant, which reduces detection sensitivity and affects practical applications.

[0003] A bipolar electrode (BPE) is a novel type of electrode, first proposed in 2000. It is a conductor placed in an electrolyte solution and not directly connected to an external power source. When a sufficiently strong positive or negative voltage is applied between the ionic phases at both ends of the bipolar electrode, the potential difference between the bipolar electrode and the solution causes a Faraday reaction at both ends of the BPE. Based on whether the electrolyte solutions at both ends of the bipolar electrode are connected, they can be divided into open-cell BPEs (o-BPE) and closed-cell BPEs (c-BPE). In an o-BPE, the electrolyte solutions at both ends are in the same reaction space. In a c-BPE, the electrolyte solutions at both ends are completely physically separated. Therefore, in a c-BPE system, the electrolyte solution does not act as a medium for electron transfer; all electrons are transferred by the closed-cell bipolar electrode itself and used to communicate the electrochemical reactions at both ends of the electrode. Compared to o-BPE, c-BPE has a significantly improved electron utilization rate. Furthermore, because the anode and cathode of c-BPE are physically separated, they can be configured as the detection end and signal output end, respectively. This configuration better reflects the environmental conditions of the actual analyte (such as buffer solution, pH, and analyte concentration), thus aligning with practical applications of the sensor. The anode and cathode of c-BPE are connected to two driving electrodes via different electrolytes. Due to the advantage of physical separation of the anode and cathode, mutual interference between redox processes can be effectively avoided. ECL based on BPE provides a portable, simple, low-background, fast-response, and wide-range analytical method.

[0004] Safflower, first recorded in the *Kaibao Materia Medica*, is also known as safflower, grass safflower, and thorn safflower. It is the tubular dried product of the herbaceous plant *Carthamus tinctorius*, belonging to the genus *Carthamus* in the Asteraceae family. It possesses the effects of promoting blood circulation, regulating menstruation, removing blood stasis, relieving pain, and calming the mind. Its dried flowers are listed as a medicinal material in the *Chinese Pharmacopoeia* (2020 edition). Traditionally, the petals of dried safflower are used in medicine, indicating a long history of medicinal use. Safflower mainly contains volatile oils and flavonoids. Flavonoids are the main active ingredients of safflower, including kaempferol, hydroxysafflower yellow A, quercetin, and rutin. Among these, the *Chinese Pharmacopoeia* lists kaempferol and hydroxysafflower yellow A as quality indicator components of safflower. The determination of the content of active ingredients in safflower is of great significance for studying the clinical efficacy, pharmacological effects, quality control, and resource development of drugs. Therefore, developing a rapid, sensitive, and on-site detection method for samples will undoubtedly promote the development of drug analysis and research.

[0005] Kaempferol (KA) is a flavonoid compound mainly found in the active components of safflower and has significant medicinal value in reducing cancer, atherosclerosis, and cardiovascular diseases. However, excessive KA is inherently toxic, making the development of highly sensitive detection methods necessary and important. Currently, some effective detection methods exist, such as capillary electrophoresis, chromatography, thin-layer chromatography (TLC), and spectrophotometry, but these methods suffer from low sensitivity, high cost, and long processing times. Bipolar electrochemiluminescence analysis is more convenient and economical than the aforementioned methods, indicating that developing a highly sensitive and stable bipolar electrochemiluminescence method for the determination of KA has great application value. Furthermore, the bipolar electrochemiluminescence method has advantages such as simple pretreatment steps, high sensitivity, and wide applicability, and holds promise for achieving rapid and accurate detection of KA.

[0006] This invention focuses on kaempferol, a flavonoid reducing agent in safflower, and constructs a novel bipolar electrochemiluminescence sensor based on a split magnetic adsorption device for the rapid and highly sensitive detection of kaempferol. The performance of the detection method in terms of detection range, sensitivity, repeatability, and long-term stability is also investigated. Summary of the Invention

[0007] The purpose of this invention is to address existing problems by providing a closed, split-type magnetic bipolar electrochemiluminescence sensor and its application in the detection of kaempferol in safflower.

[0008] This invention is achieved through the following technical solution: A closed, split-type magnetically attracted bipolar electrochemiluminescence sensor, comprising three parts: an electrode cover plate, a detection chamber, and a signal chamber; The detection chamber and the signal chamber are physically isolated and electrochemically coupled through a common bipolar electrode (BPE). The bottom of the detection chamber has an electrode connection hole with a diameter of 3.2 mm. A conductive platinum sheet is fixed at the top of the electrode connection hole by curing acrylic acid under UV conditions for 1 min. A polyimide film (PI) with a hole in the middle is covered on the conductive platinum sheet. The detection chamber can accommodate a Ni@NdFeB magnet to realize the magnetic stirring function. The detection chamber is also provided with a sample inlet 1 for injecting the test liquid. The top of the signal chamber is fixed with a Ni@NdFeB magnet, the bottom is encapsulated with quartz glass, and the signal chamber is provided with an injection port 2 for injecting luminescent liquid.

[0009] Furthermore, the luminescent liquid is Ru(bpy)3. 2+ A mixture of (ruthenium terpyridine) and TPrA@Au NPs, wherein Ru(bpy)3 2+The concentration ratio with TPrA@Au NPs was 1:50.

[0010] Furthermore, the Ru(bpy)3 2+ The concentration was 0.125 mmol·L⁻¹ -1 The solvent is ultrapure water.

[0011] Furthermore, the TPrA@Au NPs were prepared by a one-pot method, specifically: 17.5 mL of ultrapure water was heated to boiling on a heated magnetic stirrer, and 1 mL of 0.1 mol·L⁻¹ NPs was rapidly injected while stirring at 1000 rpm. -1 The concentration of TPrA in 200 μL was 22.7 mmol·L⁻¹. -1 The HAuCl4 injection operation was completed within 10 s (rapid injection ensures that the two reagents are instantly and uniformly distributed at high temperature, inhibits side reactions (such as the oxidative decomposition of TPRA) and controls the nucleation rate of nanoparticles), and the solution was heated until the color of the solution turned grayish-blue, then purple, and finally red. Finally, the prepared gold nanosol was purified by dialysis at MW: 3500 for 48 h and stored at 4°C in the dark for further use.

[0012] Furthermore, the sensing element of the sensor is prepared by designing a sketch of the sensor device using Solidworks software, printing the design drawing using a 3D printer, cleaning it, and then curing it a second time using a UV curing machine, thus preparing the required sensing element.

[0013] Furthermore, the polyimide film (PI) serves both as insulation and as a secondary fixation of the platinum sheet.

[0014] The application of a closed, split-type magnetic bipolar electrochemiluminescence sensor in the detection of kaempferol in safflower includes the following steps: (1) Safflower extract is injected into the sample inlet 1 of the detection chamber as the test solution, and luminescent liquid is injected into the sample inlet 2 of the signal chamber; (2) Place the Ni@NdFeB magnet in the detection chamber and stir the test solution at 600 rpm on a magnetic stirrer; (3) Connect the electrochemiluminescence analysis system and set the instrument parameters: the potential scan rate is 100 mV / s and the photomultiplier tube bias voltage is 700 V; (4) An external voltage is applied between the anode and the cathode to trigger the oxidation reaction of the anode in the detection chamber and the reduction reaction of the cathode in the signal chamber. The concentration of kaempferol in safflower is detected by detecting the change in ECL intensity in the signal chamber. The ECL intensity is proportional to the KA concentration.

[0015] Furthermore, the safflower extract refers to the kaempferol extract from safflower obtained after separation by silica gel column chromatography, and the silica gel column chromatography is a pretreatment step for the test solution.

[0016] Furthermore, the external voltage mentioned in step (4) is a cyclic scanning voltage of 1.3~3.0 V.

[0017] Furthermore, the chemical reactions that occur during the detection process include: Anode reaction in the detection chamber: ; BPE cathode reaction: ; BPE Anode Reaction: ; ; ; ; ; Cathode reaction in signal chamber: ; The present invention has the following advantages over the prior art: Based on the electrochemical properties of kaempferol, this invention aims to construct a novel, rapid, and efficient bipolar electrochemiluminescence sensor for the detection of kaempferol. Under the action of a driving voltage, kaempferol undergoes an electrochemical oxidation reaction on the anode surface of the detection chamber, promoting the oxygen reduction reaction at the cathode end of the bipolar electrode. This results in an increase in the number of electrons accumulated on the anode surface of the bipolar electrode, generating a Faraday current that flows through the bipolar electrode and triggers Ru(bpy)3 in the signal chamber. 2+ The electrochemiluminescence behavior of / TPrA@Au NPs was demonstrated, with the electrochemiluminescence intensity directly proportional to the concentration of kaempferol, thus enabling the detection of kaempferol. Using tripropylamine-functionalized gold nanoparticles as a co-reactant significantly improved the electrochemiluminescence efficiency. Under optimal experimental conditions, a bipolar electrochemiluminescence sensor for detecting kaempferol was obtained, with a linear range of 0.005–0.8 mmol·L⁻¹. -1 The limit of detection (LOD) is 4 μmol·L⁻¹. -1 (S / N = 3). The sensor constructed in this invention has advantages such as low cost, wide linear range, low detection limit, high sensitivity, good stability and good reproducibility, making it a novel method for detecting kaempferol with extremely high application potential. Attached Figure Description

[0018] Figure 1 A sketch of the BPE-ECL sensor device; Figure 2 Schematic diagram of the split-type magnetic BPE-ECL device; Figure 3 A schematic diagram illustrating the principle of a split-type magnetic BPE-ECL sensor for detecting KA; Figure 4 A represents the UV-Vis spectrum of TPrA@Au NPs; Figure 4 B represents the ECL curves of c-BPE-ECL systems with different co-reactants in the detection chamber containing 0.01 mM KA; Figure 4 C represents the CV curves of TPrA@Au NPs and TPrA; (D) EIS curve (the inset in the lower right corner is the equivalent circuit diagram); Figure 5 Comparison of the resistance values ​​of Ni@NdFeB with commonly used laboratory electrodes (Pt, Au, SPE, GCE); Figure 6 A shows the ECL-potential and CV curves in 0.8 mM KA, with a PMT voltage of 600V; Figure 6 B represents the ECL response curves of different batches of Ni@NdFeB; Figure 6 C represents the ECL response curve of Ni@NdFeB after 7 magnetic attraction cycles; Figure 6 D represents the stability study of Ni@NdFeB as an electrode, Ru(bpy)3 2+ / TPrA@Au NPs are used as the ECL system; Figure 7 For Ru(bpy)3 2+ ECL mechanism diagram of / TPrA@Au NPs; Figure 8 A is 0.05 mmol·L -1 The effect of KA on the ECL response intensity in different electrolyte solvents (n = 3); Figure 8 B is 0.05 mmol·L -1 Effect of KA on the ECL response intensity of hydrochloric acid solutions at different pH values ​​in the cathode cell (n=3); Figure 8 C represents the concentration of 0.125 mmol·L⁻¹ in the anolyte signal chamber at different pH values. -1 Ru(bpy)3 2+ and 6.25 mmol·L -1 The ECL response intensity of the anode when TPrA@Au NPs are in PBS (n = 3); Figure 8D represents the optimization for different CV scan rates (n = 3); Figure 9 A shows the ECL signal response at different KA concentrations; Figure 9 B is a linear calibration plot between the logarithm of KA concentration and the ECL signal response; Figure 10 A represents the addition of interfering substances BSA, Glu, Gly, glu, and Na. + K + Mg 2+ Interference immunity test of BPE-ECL sensor; Figure 10 B represents 10 low, medium, and high concentrations (0.005, 0.05, and 0.5 mmol·L⁻¹) tested in parallel. -1 The reproducibility of the sensor; Figure 10 C represents the storage of the successfully fabricated sensor for 9 days, with tests conducted every 2 days to investigate the storage stability of the BPE-ECL sensor. Detailed Implementation

[0019] To further explain the present invention, the following specific embodiments are described.

[0020] Instruments and reagents Table 1 Instruments ; Table 2 Reagents ; All the chemical reagents listed above are of analytical grade and can be used without further purification.

[0021] Example 1: 1. Preparation of TPrA@Au NPs TPrA@Au NPs were synthesized using a one-pot method. In short, 17.5 mL of ultrapure water was heated to boiling on a heated magnetic stirrer, and 1 mL of TPrA (0.1 mol·L⁻¹) was rapidly injected under vigorous stirring. -1 ) and 200 μL of HAuCl4 (22.7 mmol·L) -1 After heating for a few minutes, the solution color rapidly changed to grayish-blue, then purple, and finally red, indicating the formation of TPrA@Au NPs. Finally, the prepared gold nanosol (MW: 3500) was purified by dialyzing for 48 h and stored at 4°C in the dark for further use.

[0022] 2. Design and Working Principle of Split-Type Magnetic Adsorption Bipolar Electrochemiluminescence Sensor The sketch of the BPE-ECL sensor device used in this invention was designed using Solidworks software, as shown below. Figure 1 As shown in A and B, the design drawings are printed using a laboratory 3D printer, cleaned, and then cured a second time using a UV curing machine to produce the required sensing element.

[0023] like Figure 2 As shown, the BPE-ECL sensor consists of three parts: an electrode cover, a detection chamber, and a signal chamber. The bottom of the detection chamber has a 3.2 mm diameter electrode connection hole. An acrylic acid film, cured under UV conditions for 1 min, is used to fix a conductive platinum sheet above the hole. A perforated polyimide (PI) film is then placed on top of the platinum sheet, serving as insulation and secondary fixation for the platinum sheet. The detection chamber can perform multiple functions, including target detection and magnetic stirring. To mix the analyte, Ni@NdFeB is simply placed in the detection chamber and stirred using a magnetic stirrer. The signal chamber has Ni@NdFeB fixed at the top and encapsulated at the bottom with quartz glass. During detection, the analyte is injected into injection port 1, and the luminescent liquid is injected into injection port 2. The system is then connected to an electrochemiluminescence analysis system, and the instrument parameters are set as follows: potential scan rate of 100 mV / s and photomultiplier tube bias voltage of 700 V.

[0024] like Figure 3 As shown, the split-type magnetic BPE-ECL sensor consists of two chambers: a detection chamber with an anode and a signal chamber with a cathode. These chambers are physically isolated and electrochemically coupled through a common BPE. Applying an external voltage between the anode and cathode triggers electrochemical reactions in both chambers: oxidation at the anode (detection chamber) and reduction at the cathode (signal chamber). The BPE remains charge-neutral in both chambers, acting as a reaction mediator at both ends: reduction occurs on the BPE in the detection chamber, and oxidation occurs in the signal chamber. For drug sensing analysis, KA is detected by changes in the ECL signal. In the detection chamber, a solution of the KA analyte is added; under the applied voltage, KA is enriched on the anode. In the signal chamber, Ru(bpy)3 is added... 2+ A mixture of KA and TPrA@Au NPs. Under the influence of potential, KA undergoes further electrochemical oxidation on the anode surface of the detection chamber. The higher the KA concentration, the higher the degree of oxidation at the anode, which promotes the oxygen reduction reaction (ORR) at the cathode of the BPE. This results in an increasing accumulation of electrons on the anode surface of the BPE, generating a Faraday current that flows through the BPE and triggers Ru(bpy)3 in the signal chamber (see the overall bipolar electrochemiluminescence reaction equation below). 2+ / TPrA@AuNPs oxidation, ECL intensity is proportional to KA concentration. This detection method can achieve a high signal-to-background ratio because ECL is only generated when current flows out of the detection chamber.

[0025] Anode reaction in the detection chamber: ; BPE cathode reaction: ; BPE Anode Reaction: ; ; ; ; ; Cathode reaction in signal chamber: ; 3. Results and Discussion 3.1 Materials and Characterization Tripropylamine-functionalized gold nanoparticles were synthesized via the reduction of chloroauric acid with tripropylamine. First, the optical properties of TPRA@Au NPs were investigated, such as… Figure 4 As shown in Figure A, TPrA@Au NPs exhibit a maximum absorption peak at 530 nm, which is largely consistent with previously reported results. The performance of TPrA@Au NPs in enhancing electrochemiluminescence behavior was then investigated. Figure 4 As shown in B, when only the luminescent substance Ru(bpy)3 is present... 2+ ECL behavior is absent when TPrA is present; the ECL behavior is relatively weak when TPrA is present; the addition of the improved co-reactant TPrA@Au NPs significantly enhances the ECL strength. These results indicate that TPrA@Au NPs can act as a co-reactant for Ru(bpy)3 2+ The co-reactant helps to improve the intensity of electrochemiluminescence.

[0026] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) are representative and effective electrochemical techniques for characterizing changes in electrode surface features. A screen-printed electrode (SPE) was used in an atmosphere containing 5.0 mM [Fe(CN)6]. 3- / 4- The role of AuNPs in the electrochemiluminescence behavior was investigated by CV and EIS in 0.1M KCl solution. Figure 4 As shown in Figure C, when the excitation potential is -0.3 to 0.8 V, TPrA exhibits a weaker redox reaction compared to the bare electrode. When TPrA@Au NPs are added, [Fe(CN)6]... 3- / 4- The significantly enhanced redox reaction indicates that AuNPs have a significant promoting effect on the oxidation of TPrA, possibly due to their excellent redox activity and conductivity. Figure 4 As shown in Figure D, the Nyquist plot consists of a semicircular portion in the high-frequency range and a linear portion in the low-frequency range. The inset shows the equivalent circuit diagram of the Randle model. The equivalent circuit includes the electron transfer resistance (R). ct Solution resistance (R) s ), double-layer capacitor (C dl The resistance (W) and Warburg impedance (W) were observed when TPrA was added, compared to the bare electrode. This is because TPrA readily oxidizes to TPrA· at a certain potential on the electrode surface. + The generation of cations accelerates electron transfer; the resistance decreases significantly after the addition of TPrA@Au NPs, which is due to the large specific surface area, excellent electrical conductivity and electrocatalytic activity of the nanoparticles.

[0027] 3.2 Morphology and Performance Study of Ni@NdFeB Electrode Nickel-plated neodymium iron boron (Ni@NdFeB) is a common and inexpensive magnetic material widely used in electronics, electrical machinery, medical devices, aerospace, and other fields. Inspired by this, this work introduces Ni@NdFeB to fabricate conductive components. Figure 5 As shown, the resistance values ​​of Ni@NdFeB and commonly used laboratory electrodes (Pt, Au, SPE, GCE electrodes) were measured using a DEM P0 laboratory multimeter. The resistance value of Ni@NdFeB and Pt electrodes was as low as 0.2 Ω. However, laboratory Pt electrodes are expensive. In comparison, Ni@NdFeB has the advantages of being easy to use, low cost, and disposable.

[0028] like Figure 6As shown in Figure A, the ECL and electrochemical performance of Ni@NdFeB as a BPE were investigated, and the optimal driving voltage range in the electrochemiluminescence system was further examined. With other parameters remaining constant, the effect of driving voltage from 1.3 V to 3.0 V on ECL intensity was studied. With increasing driving voltage, the ECL intensity gradually increased, reaching its maximum after 3.0 V, indicating that 3.0 V is the optimal driving voltage. Furthermore, the redox peak of the CV curve corresponds to the oxidation reaction of KA at the anode in the detection chamber, and the driving voltage for subsequent experiments was set to 1.3–3.0 V. With increasing electrode diameter and length, the internal resistance of the electrode also increases, and the luminescence intensity gradually stabilizes. Considering experimental costs, Ni@NdFeB with a diameter of 2 mm and a length of 15 mm was used for subsequent experiments. Given that Ni@NdFeB is not a commercially available electrode for laboratory use and faces many challenges such as complex conditions and factors in actual testing, its stability and repeatability were tested. KA was measured using seven different batches of Ni@NdFeB to evaluate its batch-to-batch reproducibility. Figure 6 As shown in Figure B, the relative standard deviation of the seven electrodes is less than 4% (n = 3), demonstrating that Ni@NdFeB exhibits good batch-to-batch reproducibility. Figure 6 As shown in Figure C, the relative standard deviation of 7 magnetic attraction cycles using the same electrode is less than 4% (n = 3), indicating that the number of magnetic attraction cycles has little effect on the ECL strength. Finally, as... Figure 6 As shown in Figure D, Ni@NdFeB was used as the reaction electrode. The detection end contained 0.1 M KCl PBS, and the signal end used Ru(bpy)3. 2+ The / TPrA@AuNPs system underwent 25 consecutive tests, and the ECL signal intensity showed no significant attenuation, with an RSD of less than 3% (n = 25), indicating good stability. These results fully demonstrate the feasibility of Ni@NdFeB as a BPE sensing electrode, allowing for further testing experiments.

[0029] 3.3 Ru(bpy)3 2+ ECL mechanism of / TPrA@Au NPs In this work, the ECL luminescent material is Ru(bpy)3. 2+ The co-reactants are TPRA@Au NPs. Currently, Ru(bpy)3 2+ The mechanism of the -TPrA ECL system can be summarized as a "redox" co-reaction mechanism. During the electrochemiluminescence reaction, the anode Ru(bpy)3... 2+ The ECL mechanism of / TPrA@Au NPs is as follows: Figure 7As shown, in electrochemiluminescence experiments conducted at anodic potentials with excitation voltages of 1.3–3.0 V, the following reaction occurs. First, Ru(bpy)3… 2+ Both TPR and TPrA lose electrons to form Ru(bpy)3 in its oxidized state. 3+ and TPRA· + Subsequently, TPRA· + The loss of a proton generates TPrA·, which has a strong reducing ability. TPrA· acts as a co-reactant in the luminescent system, reducing high-valence Ru(bpy)3. 3+ Generate [Ru(bpy)3] 2+ Finally, due to [Ru(bpy)3 2+ *In the excited state, it is unstable. The outer electrons return to the ground state, emit photons, and form a stable Ru(bpy)3. 2+ Under anodic potential excitation, Ru(bpy)3 2+ This cycle continues, constantly releasing photons. The presence of AuNPs significantly amplifies the signal response of electrochemical and electrochemiluminescence processes. This is due to the excellent electrocatalytic activity of AuNPs, which accelerates the reaction of TPRA and Ru(bpy)3. 2+ The oxidation reaction improves the electron transport efficiency at the electrode interface and increases the intensity of the ECL response signal.

[0030] 3.4 Optimization of Experimental Conditions Before applying the split-type magnetic BPE-ECL sensor to the detection of actual targets, different experimental conditions need to be optimized to obtain the best sensing performance. Here, we evaluated the influence of key factors such as electrolyte solvent type, pH values ​​of the cathode detection chamber and anode signal chamber, and CV scan rate on the ECL response signal. For the detection of KA, different solvents and their pH values ​​have a significant impact on the ECL signal. Therefore, at 0.1 mol·L⁻¹, -1 0.05 mmol·L⁻¹ of nitric acid, PBS, acetic acid, phosphoric acid, sulfuric acid, and hydrochloric acid. -1 The ECL response signal of KA was investigated. For example... Figure 8 As shown in Figure A, the experimental results indicate that the ECL response signal of KA is the largest in hydrochloric acid solution; therefore, hydrochloric acid was chosen as the solvent for KA in this experiment. Subsequently, in order to achieve high-sensitivity detection of KA by the c-BPE-ECL sensing platform, the ECL behavior of KA in the cathode detection chamber in hydrochloric acid solutions at different pH values ​​(0.5~3.5) was studied. Figure 8As shown in Figure B, the ECL response signal gradually increases with increasing pH of the hydrochloric acid solution, reaching its maximum at pH 2.5. With further increases in pH, the ECL signal significantly decreases; therefore, the optimal pH for hydrochloric acid in the cathode detection chamber is 2.5. The effect of different pH values ​​in the anodic signal chamber on the ECL signal was then investigated. Figure 8 As shown in Figure C, when the pH value increases from 6.5 to 8.0, Ru(bpy)3 2+ The ECL response signal intensity increased, but decreased sharply when the pH increased from 8.0 to 9.0. This phenomenon may be because at lower pH conditions, free radical cations are less likely to deprotonate and form free radical intermediates, resulting in a lower ECL response signal. Conversely, at higher pH conditions, Ru(bpy)3... 2+ Will with OH − A competitive reaction can also lead to a lower ECL signal.

[0031] 3.5 Performance Study of Bipolar Electrochemiluminescence Sensor for Detecting KA The relationship between KA concentration and ECL response signal was investigated using the constructed BPE-ECL sensor under optimal experimental conditions. Figure 9 As shown in Figures A and B, the ECL response signal intensity is positively correlated with the logarithm of the KA concentration. After linear fitting, the linear equation is I = 3044.67log C. KA +7782.02 ( R 2 = 0.9980), with a linear range of 0.005–0.8 mmol·L⁻¹. -1 The limit of detection (LOD) is 4 μmol·L⁻¹. -1 (3σ / k, where k is the slope of the constructed standard curve and σ is the standard deviation of the 9 blank samples) (S / N = 3). Figure 9 Inset B shows that the ECL signal intensity increases with increasing KA concentration in the sample solution. This is attributed to the higher KA concentration, the greater the degree of oxidation at the anode in the detection chamber, which promotes the ORR reaction at the BPE cathode. This results in more electrons accumulating on the surface of the BPE anode electrode, further increasing the Faraday current through the c-BPE and thus enhancing the ECL signal intensity. The results indicate that this BPE-ECL sensor exhibits good KA detection performance.

[0032] Table 3 summarizes the sensing performance of currently reported KA detection methods and the bipolar electrochemiluminescence sensor constructed in this study. According to the information in the table, compared to other detection methods, our constructed bipolar electrochemiluminescence sensor still exhibits quite good performance in terms of sensitivity and response detection range without any modification to enhance the sensitizing material.

[0033] Table 3 compares the prepared bipolar electrochemiluminescence sensor with other reported detection methods in the detection of KA. ; 3.6 Evaluation of the sensor's anti-interference, repeatability, and stability Furthermore, the performance of the BPE-ECL sensor in testing KA was verified through ECL testing, and the anti-interference, reproducibility, and storage stability of the constructed sensor under optimal experimental conditions were investigated. First, the impact of potential impurities in the sample on KA was tested, such as... Figure 10 As shown in A, 0.1 mmol·L -1 A certain concentration of interfering substances was added to KA, and the ECL response of KA was measured. The results showed that 200 times the concentration of bovine serum albumin (BSA), glutamate (Glu), glycine (Gly), glucose (glu), and Na+... + K + Mg 2+ The difference between having added interfering substances and not having added interfering substances is not significant. For example... Figure 10 As shown in Figure B, the reproducibility of the BPE-ECL sensor was investigated at low, medium, and high concentrations (0.005, 0.05, and 0.5 mmol·L⁻¹). -1 The KA of the BPE-ECL sensor was tested 10 times. The results showed that the response values ​​of different batches of the sensor did not vary significantly, with RSDs of 2.98%, 4.78%, and 2.22%, respectively. Subsequently, storage stability tests were also conducted on the BPE-ECL sensor. Figure 10 As shown in C, the prepared sensor was stored at 4°C for 0.8 mmol·L⁻¹. -1 The KA (kcal / caryophylla) content was measured, and ECL (electrochemical coefficient) tests were performed every two days. The results showed that the sensor still maintained a good ECL response signal after 9 days of storage, with a relative standard deviation of only 2.63%. These data indicate that the BPE-ECL sensor possesses good anti-interference capabilities, reproducibility, and storage stability, and is expected to be applied to the detection of KA, an active substance in safflower.

[0034] The above test was conducted in a solution containing 6.25 mmol·L⁻¹ -1 TPrA@Au NPs were analyzed in PBS solution at pH 8.0.

[0035] 4. Conclusion In summary, this invention constructs a low-cost, portable BPE-ECL sensing platform based on Ni@NdFeB, and significantly improves the electrochemiluminescence efficiency by using tripropylamine-functionalized gold nanoparticles as a co-reactant. This BPE-ECL device successfully achieves highly sensitive detection of KA, and within a certain KA concentration range, the ECL response signal exhibits a good linear relationship with the KA concentration. The linear range is 0.005–0.8 mmol·L⁻¹. -1 The limit of detection (LOD) is 4 μmol·L⁻¹. -1 (S / N = 3). The constructed sensor possesses advantages such as low cost, wide linear range, low detection limit, high sensitivity, good stability, and good reproducibility, making it a novel method for detecting kaempferol with extremely high application potential. In the near future, we will utilize this Ni@NdFeB-based BPE-ECL sensing platform to achieve high-throughput detection.

[0036] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A closed, split-type magnetically attracted bipolar electrochemiluminescence sensor, characterized in that, The sensor consists of three parts: an electrode cover plate, a detection chamber, and a signal chamber. The detection chamber and the signal chamber are physically isolated and electrochemically coupled through a common bipolar electrode. The bottom of the detection chamber has an electrode connection hole with a diameter of 3.2 mm. A conductive platinum sheet is fixed at the top of the electrode connection hole by curing acrylic acid under UV conditions for 1 min. A polyimide film with a hole in the middle is covered on the conductive platinum sheet. The detection chamber can accommodate a Ni@NdFeB magnet to realize the magnetic stirring function. The detection chamber is also provided with a sample inlet 1 for injecting the test liquid. The top of the signal chamber is fixed with a Ni@NdFeB magnet, the bottom is encapsulated with quartz glass, and the signal chamber is provided with an injection port 2 for injecting luminescent liquid.

2. The enclosed, split-type magnetic bipolar electrochemiluminescence sensor according to claim 1, characterized in that, The luminescent liquid is Ru(bpy)3 2+ A mixture of Ru(bpy)3 and TPrA@Au NPs 2+ The concentration ratio with TPrA@Au NPs was 1:

50.

3. A closed, split-type magnetically attracted bipolar electrochemiluminescence sensor according to claim 2, characterized in that, The Ru(bpy)3 2+ The concentration was 0.125 mmol·L⁻¹ -1 The solvent is ultrapure water.

4. A closed, split-type magnetically attracted bipolar electrochemiluminescence sensor according to claim 2, characterized in that, The TPrA@Au NPs were prepared by a one-pot method, specifically: 17.5 mL of ultrapure water was heated to boiling on a heated magnetic stirrer, and 1 mL of 0.1 mol·L⁻¹ NPs was rapidly injected while stirring at 1000 rpm. -1 The concentration of TPrA in 200 μL was 22.7 mmol·L⁻¹. -1 The HAuCl4 injection operation was completed within 10 s. The solution was heated until the color turned grayish-blue, then purple, and finally red. Finally, the prepared gold nanosol was purified by dialysis at MW: 3500 for 48 h and stored at 4°C in the dark for further use.

5. The enclosed, split-type magnetic bipolar electrochemiluminescence sensor according to claim 1, characterized in that, The sensing element of the sensor is prepared by designing a sketch of the sensor device using Solidworks software, printing the design drawing using a 3D printer, cleaning it, and then curing it a second time using a UV curing machine to prepare the required sensing element.

6. The enclosed, split-type magnetic bipolar electrochemiluminescence sensor according to claim 1, characterized in that, The polyimide film serves both as insulation and as a secondary fixation of the platinum sheet.

7. The application of a closed-type split magnetic bipolar electrochemiluminescence sensor as described in any one of claims 1 to 6 in the detection of kaempferol in safflower, characterized in that, Includes the following steps: (1) Safflower extract is injected into the sample inlet 1 of the detection chamber as the test solution, and luminescent liquid is injected into the sample inlet 2 of the signal chamber; (2) Place the Ni@NdFeB magnet in the detection chamber and stir the test solution at 600 rpm on a magnetic stirrer; (3) Connect the electrochemiluminescence analysis system and set the instrument parameters: the potential scan rate is 100 mV / s and the photomultiplier tube bias voltage is 700 V; (4) An external voltage is applied between the anode and the cathode to trigger the oxidation reaction of the anode in the detection chamber and the reduction reaction of the cathode in the signal chamber. The concentration of kaempferol in safflower is detected by detecting the change in ECL intensity in the signal chamber. The ECL intensity is proportional to the KA concentration.

8. The application of the closed-type split magnetic bipolar electrochemiluminescence sensor according to claim 7 in the detection of kaempferol in safflower, characterized in that, The external voltage mentioned in step (4) is a cyclic scanning voltage of 1.3~3.0 V.

9. The application of the closed-type split magnetic bipolar electrochemiluminescence sensor according to claim 7 in the detection of kaempferol in safflower, characterized in that, The chemical reactions that occur during the detection process include: Anode reaction in the detection chamber: ； BPE cathode reaction: ； BPE Anode Reaction: ； ； ； ； ； Cathode reaction in signal chamber: 。

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