A method for constructing an electrochemiluminescence system based on natural dyes and its application

Abstract

The present invention discloses a construction method and application of an electrochemiluminescence system based on natural dyes, using natural dyes as ECL emitters for the detection of target substances; correlating the statistically obtained luminescence intensity with different natural dye emitters and electrolyte types to obtain the optimal luminescence conditions; different from traditional commercial ECL emitters, such ECL emitters belong to natural dyes, exist in organisms such as the human body and bacteria, have the characteristics of being environmentally friendly and easily available, and having natural metabolic pathways, conform to the green development strategy, and therefore have higher potential commercial value. The ECL device used in the present invention is simple and easy to operate, and can conveniently detect the change trend of light intensity with the concentration of reactants; the reaction initial conditions, speed, and process can be controlled by adjusting the potential using the ECL detection method, facilitating in-situ and on-site analysis; it is applicable to the detection of metal ions and the analysis of H2O2, etc.

Description

Technical Field

[0001] The invention belongs to the technical field of physical and chemical analysis, and in particular relates to a construction method and application of an electrochemiluminescence system based on natural dyes. Background Art

[0002] Electrochemiluminescence (ECL) is a type of light radiation generated by applying a certain voltage to the electrode to cause a chemical reaction between the electrode reaction products or between the electrode reaction products and a component in the solution. ECL has the advantages of both electrochemical and chemiluminescence methods, and has the advantages of high sensitivity, extremely low detection limit, wide linear range, simple instrumentation, convenient operation, and easy automation. ECL technology is a new detection technology that not only provides luminescence intensity and electrochemical signals, but also provides more information in optical detection and imaging. Current commercial applications mainly rely on the traditional system of ruthenium bipyridine and its derivatives that respond to positive potential. Although many organic dyes, including polycyclic aromatic hydrocarbons, heterocyclic compounds, including the boron-dipyrromethene (BODIPY) series, have shown their outstanding potential in ECL, due to their low solubility in aqueous solution, or the instability of reaction transition states and free radical intermediates, and the narrow potential window of the working electrode in aqueous solution, the ECL of very few molecular luminophores can be reproduced in the aqueous phase.

[0003] In 1927, Dufford et al. observed the ECL phenomenon when electrolyzing Grignard reagent in anhydrous ether. In 1929, Harvey discovered luminescence near the electrode when electrolyzing alkaline luminol aqueous solution. Later in the 1960s and 1970s, when people used ECL as a tool to study compounds, complexes and cluster molecules with new photochemical and electrochemical properties, they gradually realized that this work was very helpful in clarifying the mechanism of chemiluminescence, and the study of the ECL phenomenon has since received attention. Since the 1980s, with the improvement of various electrodes and instrument systems and the discovery of various new systems, the study of the ECL reaction mechanism has become increasingly mature. As a highly sensitive and highly selective analytical method, ECL has attracted increasing attention and has become an effective detection method in analytical chemistry. However, most of the commercial ECL luminophores (such as pyridines, acridinium esters, etc.) are artificially synthesized products. Unlike natural dyes in the field of fluorescence detection, there are few reports on ECL-active natural dye luminophores. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a method for constructing an electrochemiluminescence system based on natural dyes and its application.

[0005] The objective of the present invention is achieved through the following technical scheme: a method for constructing an organic phase electrochemiluminescence system based on natural dyes, dispersing the natural dye luminophore in an organic phase system electrolyte or modifying it on a bare electrode, and directly inducing the natural dye luminophore to produce ECL; the natural dye luminophore is a linear tetrapyrrole analogue.

[0006] Furthermore, the organic phase system electrolyte comprises acetonitrile electrolyte or dichloromethane electrolyte.

[0007] Furthermore, the concentration of the natural dye luminophore is 0-3mM; the concentration range of the electrolyte is 0-0.3M.

[0008] Furthermore, the natural dye luminophore comprises phycocyanin, phycoerythrin, biliverdin, mesobiliredin, bilirubin, mesobilirubin, urobilinogen, sterobilin, bilirubin or the like.

[0009] A method for constructing an aqueous electrochemiluminescence system based on natural dyes, wherein the natural dye luminophore is dispersed in an aqueous system electrolyte or modified on a bare electrode, and then the electrode is modified with tetraoctylammonium bromide to induce the natural dye luminophore to produce ECL; the natural dye luminophore is a linear tetrapyrrole analogue.

[0010] Furthermore, the aqueous system buffer electrolyte includes HEPES buffer electrolyte, PBS buffer electrolyte, Tris-HCl buffer electrolyte, CH3COOH / CH3COONa buffer electrolyte or H3BO3 / Na2B4O7 buffer electrolyte.

[0011] Furthermore, the concentration of the aqueous system buffer electrolyte is 1-50 mM and the corresponding pH range is 6-9, the electrolyte concentration is 0-0.3 M; the concentration of the natural dye luminophore is 0-3 mM; and the concentration of tetraoctylammonium bromide is 0-25 mM.

[0012] Furthermore, the natural dye luminophore comprises phycocyanin, phycoerythrin, biliverdin, mesobiliredin, bilirubin, mesobilirubin, urobilinogen, sterobilin, bilirubin or the like.

[0013] A method for detecting the concentration of a substance in an electrochemiluminescent system based on a natural dye comprises the following steps:

[0014] Step 1: constructing an electrochemiluminescence system based on the construction method of claim 1 or 6; wherein a solution of a substance to be tested is added to an electrolyte;

[0015] Step 2: Use an ECL luminometer to record the luminescence intensity of the natural dye luminophore, and perform numerical fitting to obtain a mathematical model based on the luminescence intensity of the natural dye luminophore corresponding to the solutions of the test substance with different concentrations; the concentration of the test substance can be calculated based on the fitted mathematical model and the luminescence intensity corresponding to the solution of the test substance.

[0016] Furthermore, the ECL luminometer parameter settings include: ECL photomultiplier tube bias voltage is -1000 to -100V, the amplification level is 3, the scanning rate is 0 to 0.3V / s, and the scanning potential is 0.5 to -2.5V.

[0017] The beneficial effects of the present invention are as follows:

[0018] (1) The present invention uses natural dyes with ECL activity as luminophores; the statistically obtained luminescence intensity is associated with the luminophore concentration and the type of buffer solution to obtain the optimal luminescence conditions; different from the current commercial luminophores, the natural dye luminophores provided by the present invention have good ECL activity, are environmentally friendly and easy to obtain, and have natural metabolic pathways, which are in line with the green development strategy and therefore have higher potential commercial and scientific research value;

[0019] (2) The ECL device used in the present invention is simple and easy to operate, and can conveniently detect the trend of light intensity changing with the concentration of the object to be detected;

[0020] (3) The ECL detection method of the present invention can control the initial conditions, speed and course of the reaction by adjusting the potential, and can conveniently perform in-situ analysis and on-site analysis;

[0021] (4) The present invention is suitable for detecting trace metal ion concentration and H2O2 analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The ECL test diagrams of natural dyes with different 0.1 mM ECL activities in acetonitrile electrolyte; (A) is phycocyanin, (B) is biliverdin, (C) is mesobililiverdin, (D) is bilirubin, (E) is mesobilirubin, (F) is urobilinogen, (G) is sterobilin, and (H) is bilirubic acid; a is the electrochemical cyclic voltammetry curve, and b is the ECL curve;

[0023] Figure 2 This is the ECL spectrum of the natural dye phycocyanin luminophore in acetonitrile electrolyte;

[0024] Figure 3 This is the ECL spectrum of the natural dye luminophore PCB in HEPES buffered electrolyte;

[0025] Figure 4These are the ECL test graphs of natural dye luminophore PCB in HEPES buffered electrolyte under nitrogen, nitrogen + H2O2, air, and oxygen atmospheres, respectively; the interpolation is a bar chart comparison of ECL intensity at -1.5V;

[0026] Figure 5 The ECL curve diagram of the luminescence intensity of the natural dye luminophore PCB in a HEPES buffered electrolyte under a nitrogen atmosphere is affected by different concentrations of H2O2; wherein (A) is a standard curve diagram, and (B) is an ECL stability test diagram;

[0027] Figure 6 The figure is an ECL curve diagram showing the change of the luminescence intensity of the natural dye luminophore PCB in the HEPES buffered electrolyte under the influence of different concentrations of copper ions; wherein, the interpolation is a standard curve diagram of the ECL intensity at -1.5V and the copper ion concentration fitted by the dynamic quenching constant model calculated according to the Stern-Volmer equation. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with specific embodiments and drawings.

[0029] The present invention discloses a method for constructing an organic phase electrochemiluminescence system based on natural dyes, which can directly induce natural dye luminophores to produce ECL using bare electrodes. Specifically, a natural dye luminophore with ECL activity is drop-coated on the electrode surface or dispersed in an organic phase system electrolyte to perform an ECL test. The electrodes include but are not limited to glassy carbon electrodes, gold electrodes, and platinum electrodes. The concentration range of the organic phase system electrolyte is 0 to 0.3 M, and the concentration range of the natural dye luminophore is 0 to 3 mM. The parameters of the ECL luminometer are set as follows: the ECL photomultiplier tube bias is -1000 to -100 V, the amplification level is 3, the scanning rate is 0 to 0.3 V / s, and the scanning potential is 0.5 to -2.5 V. The organic phase system electrolyte includes an acetonitrile electrolyte or a dichloromethane electrolyte. The natural dye luminophores with electrochemiluminescent activity are mainly phycocyanin, phycoerythrin, biliverdin, mesobililiverdin, bilirubin, mesobilirubin, urobilinogen, sterobilin, bilirubin or the like, all of which are linear tetrapyrrole analogs.

[0030] The present invention discloses a method for constructing an aqueous electrochemiluminescence system based on natural dyes. Under an electrode modified with a tetraoctylammonium bromide (TOAB) film, the natural dye luminophore can be induced to produce ECL; specifically, a natural dye luminophore with ECL activity is drop-coated on the electrode surface or dispersed in an aqueous system buffer electrolyte, and the electrode surface is modified with a TOAB film before an ECL luminescence test is performed. The electrode includes but is not limited to a glassy carbon electrode, a gold electrode, and a platinum electrode. The aqueous system buffer electrolyte includes a HEPES buffer electrolyte, a PBS buffer electrolyte, a Tris-HCl buffer electrolyte, a CH3COOH / CH3COONa buffer electrolyte, or a H3BO3 / Na2B4O7 buffer electrolyte. The aqueous system buffer electrolyte has a concentration range of 1 to 50 mM and a corresponding pH range of 6 to 9, and an electrolyte concentration range of 0 to 0.3 M. The concentration range of the natural dye luminophore is 0 to 3 mM, and the concentration range of TOAB is 0 to 25 mM. The parameters of the ECL luminometer were set as follows: ECL photomultiplier tube bias voltage was -1000 to -100 V, the amplification level was 3, the scanning rate was 0 to 0.3 V / s, and the scanning potential was 0.5 to -2 V.

[0031] The present invention adopts an ECL luminometer to perform ECL testing on the natural dye luminophore, and has application value in both organic phase system and aqueous phase system detection.

[0032] A detection application of an organic phase electrochemiluminescence system based on natural dyes comprises the following steps:

[0033] Step 1: Obtain a natural dye luminophore solution with ECL activity, solutions of the substance to be tested with different concentrations, and an organic phase system electrolyte.

[0034] Step 2: Add an organic phase system electrolyte to the electrolytic cell, then add the natural dye luminophore solution to the electrolyte, and finally add the substance to be tested to the electrolyte, so that the ECL luminescence intensity of the natural dye luminophore is correlated with the concentration of the substance to be tested.

[0035] Step 3: Immerse the bare glassy carbon electrode, platinum electrode, and Ag / AgCl electrode into the electrolyte as the working electrode, counter electrode, and reference electrode, respectively.

[0036] Step 4: Turn on the ECL luminometer and record the relationship between the luminescence intensity of the natural dye luminophore and the voltage, and the relationship between the luminescence intensity and time.

[0037] Step 5: Transfer to a computer, analyze the intensity of the natural dye luminophore when the concentration of the substance to be tested changes, and perform numerical fitting to obtain a mathematical model. The concentration of the substance to be tested can be calculated based on the fitted mathematical model and the luminescence intensity corresponding to the solution of the substance to be tested.

[0038] A detection application of an aqueous electrochemiluminescence system based on natural dyes comprises the following steps:

[0039] Step 1: Obtain a natural dye luminophore solution with ECL activity, solutions of test substances with different concentrations, and an aqueous system buffer electrolyte.

[0040] Step 2: Add an aqueous buffered electrolyte to the electrolytic cell, then add the substance to be tested to the electrolyte, and finally drop the natural dye luminophore and TOAB on the glassy carbon electrode in sequence, so that the ECL luminescence intensity of the natural dye luminophore is correlated with the concentration of the substance to be tested.

[0041] Step 3: Immerse the glassy carbon electrode, platinum electrode, and Ag / AgCl electrode modified with the luminescent body into the electrolyte as the working electrode, counter electrode, and reference electrode, respectively.

[0042] Step 4: Turn on the ECL luminometer and record the relationship between the luminescence intensity of the natural dye luminophore and the voltage, and the relationship between the luminescence intensity and time.

[0043] Step 5: Transfer to a computer, analyze the intensity of the natural dye luminophore when the concentration of the substance to be tested changes, and perform numerical fitting to obtain a mathematical model. The concentration of the substance to be tested can be calculated based on the fitted mathematical model and the luminescence intensity corresponding to the solution of the substance to be tested.

[0044] Example 1

[0045] This embodiment provides a natural dye luminophore with ECL activity, which can generate stable ECL in acetonitrile electrolyte, and the specific steps are as follows:

[0046] Step 1: Prepare 0.1 M tetrabutylammonium hexafluorophosphate in acetonitrile electrolyte.

[0047] Step 2: Various ECL-active natural dyes were dispersed in tetrabutylammonium hexafluorophosphate in acetonitrile electrolyte at a concentration of 0.1 mM.

[0048] Step 3: Use a bare glassy carbon electrode to perform an ECL test and record the ECL intensity of the ECL-active natural dye dispersed in the acetonitrile electrolyte of tetrabutylammonium hexafluorophosphate. The specific test results are as follows: Figure 1 As shown, Figure 1 Give the molecular structure formula of each natural dye. Figure 1It can be found that (A) is the ECL test of phycocyanin, with a luminescence potential of -2.2V and -0.2V, (B) is the ECL test of biliverdin, with a luminescence potential of -2.0V, -0.3V and -0.07V, (C) is the ECL test of mesobililiverdin, with a luminescence potential of -2.1V and -0.05V, (D) is the ECL test of bilirubin, with a luminescence potential of -2.0V, -0.3V and +0.1V, (E) is the ECL test of mesobilirubin, with a luminescence potential of -2.0V and -0.08V, (F) is the ECL test of urobilinogen, with a luminescence potential of -2.1V, (G) is the ECL test of sterobilin, with a luminescence potential of -2.1V, and (H) is the ECL test of bilirubic acid, with a luminescence potential of -2.0V and -0.05V.

[0049] Step 4: Use a bare glassy carbon electrode to perform ECL testing. At the same time, place filters of different wavelengths between the electrolytic cell and the ECL luminometer window. Record the ECL intensity and spectral information generated by the ECL-active natural dye luminophycocyanin (PCB) in the acetonitrile electrolyte of tetrabutylammonium hexafluorophosphate under the same instrument parameters. The specific test results are as follows: Figure 2 As shown. Figure 2 It can be found that around -0.2V ( Figure 2 a) and -2.2V ( Figure 2 b) have different luminescence spectra, with luminescence peaks at 680 nm and 640 nm, respectively, indicating that the ECL-active natural dye provided by the present invention is an ECL luminophore that produces luminescence of different wavelengths under different voltages.

[0050] In this embodiment, the detection system used is an ECL luminometer. The parameters of the ECL luminometer include an ECL photomultiplier tube bias of -1000V, amplification level of 3, a scanning rate of 0.1V / s, a scanning potential of 0.2 to -2.4V, and an acetonitrile electrolyte in the organic phase system.

[0051] Example 2

[0052] This example takes an ECL-active natural dye luminophore as an example to generate stable ECL in a HEPES buffered electrolyte. The specific steps are as follows:

[0053] Step 1: Prepare 10 mM HEPES buffered electrolyte containing 0.1 M potassium chloride.

[0054] Step 2: Use 20 μL of 1.5 mM ECL-active natural dye luminophore PCB droplet to coat a 5 mm diameter glassy carbon electrode.

[0055] Step 3: Modify the 10mM TOAB film on the glassy carbon electrode with the luminophore drop-coated, and perform ECL test. At the same time, place filters of different wavelengths between the electrolytic cell and the light window of the ECL luminometer, and record the ECL intensity and spectral information generated by the ECL-active natural dye luminophore PCB in the HEPES buffered electrolyte under the same instrument parameters. The specific test results are as follows: Figure 3 As shown. Figure 3 It can be found that the composite luminescence peaks of the ECL spectrum of PCB at around -1.5V in HEPES buffered electrolyte are at 680nm and 635nm.

[0056] In this embodiment, the detection system used is an ECL luminometer. The parameters of the ECL luminometer include an ECL photomultiplier tube bias of -1000V, an amplification level of 3, a scanning rate of 0.1V / s, a scanning potential of 0 to -1.6V, and an aqueous phase system using HEPES buffered electrolyte and a corresponding pH of 7.5.

[0057] Example 3

[0058] This embodiment takes an ECL-active natural dye luminophore as an example to investigate the ECL test and electrochemical characteristics of the natural dye luminophore PCB in air, nitrogen, oxygen atmosphere and in the presence of a co-reactant H2O2. The specific steps are as follows:

[0059] Step 1: Prepare 10 mM HEPES buffered electrolyte containing 0.1 M potassium chloride.

[0060] Step 2: Use 20 μL of 1.5 mM natural dye luminophore PCB to drop onto a 5 mm diameter glassy carbon electrode.

[0061] Step 3: Modify 20 μL of 10 mM TOAB film on the glassy carbon electrode with the luminophore drop-coated, and perform ECL tests in air, nitrogen, oxygen atmosphere and in the presence of co-reactant H2O2, and record the ECL light intensity generated by the ECL-active natural dye luminophore PCB in the HEPES buffered electrolyte under the same instrument parameters. The specific test results are as follows: Figure 4 As shown. Figure 4 It can be seen that oxygen and H2O2 are both co-reactants of PCB's ECL. When oxygen is used as a co-reactant, the ECL potential of PCB shifts slightly negatively. There is almost no ECL intensity of PCB in nitrogen atmosphere. The ECL intensity of PCB in air atmosphere is 0.2 times that in oxygen atmosphere, which is consistent with the oxygen content in the air.

[0062] In this embodiment, the detection system used is an ECL luminometer. The parameters of the ECL luminometer include an ECL photomultiplier tube bias of -700 V, amplification level of 3, a scanning rate of 0.1 V / s, a scanning potential of 0 to -1.6 V, and an aqueous phase system buffer using HEPES buffer and a corresponding pH of 7.5.

[0063] Example 4

[0064] This embodiment takes an ECL-active natural dye luminophore as an example to provide an analytical technique for measuring the change in the luminescence intensity of the natural dye luminophore PCB in a HEPES buffered electrolyte under the influence of H2O2. The specific steps are as follows:

[0065] Step 1: Prepare 10 mM HEPES buffered electrolyte containing 0.1 M potassium chloride.

[0066] Step 2: Use 20 μL of 1.5 mM natural dye luminophore PCB to drop onto a 5 mm diameter glassy carbon electrode.

[0067] Step 3: Modify the glassy carbon electrode with 20μL 10mM TOAB film on which the luminophore was drop-coated, and perform ECL test in a nitrogen atmosphere and the presence of co-reactant H2O2. Record the ECL light intensity generated by the ECL-active natural dye luminophore PCB in the HEPES buffered electrolyte under the same instrument parameters. The specific test results are shown in Figure 5.

[0068] from Figure 5 As can be seen from (A), the concentrations of H2O2 are 0.1mM, 1mM, 10mM, 25mM, 50mM, 75mM, and 100mM, respectively. When H2O2 is used as a coreactant, the maximum effective concentration is about 50mM. The linear range of detection is 0.1mM to 50mM. The fitting equation is y=102.59x-9.09, and the regression coefficient R 2 =0.99; from Figure 5 As can be seen in (B), the PCB provided by the present invention as a luminous body has good luminescence stability within 1000s, and the RSD value of the peak statistics in the figure is about 1.45%.

[0069] In this embodiment, the detection system used is an ECL luminometer. The parameters of the ECL luminometer include an ECL photomultiplier tube bias of -500V, amplification level of 3, a scanning rate of 0.1V / s, a scanning potential of 0 to -1.6V, and an aqueous phase system buffer using HEPES buffer and a corresponding pH of 7.5.

[0070] Example 5

[0071] This embodiment takes a natural dye luminophore with ECL activity as an example to provide an analytical technique for measuring the change in the luminescence intensity of the natural dye luminophore PCB in a HEPES buffer under the influence of copper ions. The specific steps are as follows:

[0072] Step 1: Prepare ECL-active natural dye luminophore solution, metal ion solution and buffered electrolyte.

[0073] Step 2: Use 20 μL of 1.5 mM natural dye luminophore PCB to drop onto a 5 mm diameter glassy carbon electrode.

[0074] Step 3: Modify the glassy carbon electrode with 20 μL 10 mM TOAB film on the drop-coated luminophore, then add copper ion solution to the HEPES buffer electrolyte, perform ECL test and record the ECL intensity under the same instrument parameters at different copper ion concentrations. Calculate the dynamic quenching constant model based on the Stern-Volmer equation to analyze the corresponding intensity change of the luminophore when the copper ion concentration changes. The specific test results are as follows: Figure 6 As shown. Figure 6 It can be found that the luminescence intensity of PCB in HEPES buffer electrolyte decreases with the increase of copper ion concentration. The copper ion concentrations are 0μM, 0.05μM, 0.1μM, 0.5μM, 1μM, 2μM, 5μM, and 10μM, respectively. The linear range of detection is 0.05μM to 10μM. The fitting equation is y=2.04x-0.07, and the regression coefficient R 2 =0.99, the static quenching constant is about K sv =2.04×10 6 M -1 Assuming the signal-to-noise ratio S / N = 3, the minimum detection limit was calculated to be approximately 0.035 μM.

[0075] In this embodiment, the detection system used is an ECL luminometer. The parameters of the ECL luminometer include an ECL photomultiplier tube bias of -1000V, amplification level of 3, a scanning rate of 0.1V / s, a scanning potential of 0 to -1.6V, and an aqueous phase system buffer using HEPES buffer and a corresponding pH of 7.5.

Claims

1. A method for constructing an organic phase electrochemiluminescence system based on natural dyes, characterized in that: The natural dye luminophore is dispersed in an organic phase system electrolyte to directly induce the natural dye luminophore to produce electrochemiluminescence (ECL); the natural dye luminophore is phycocyanin, biliverdin, mesobililiverdin, bilirubin, mesobilirubin, urobilinogen, sterobilin or bilirubic acid; the organic phase system electrolyte is an acetonitrile electrolyte of tetrabutylammonium hexafluorophosphate.

2. The method for constructing an organic phase electrochemiluminescence system based on natural dyes according to claim 1, characterized in that: The concentration of natural dye luminophore is 0~3 mM; the electrolyte concentration range is 0~0.3 M.

3. A method for constructing an aqueous electrochemiluminescence system based on natural dyes, characterized in that: After modifying the natural dye luminophore on the bare electrode, tetraoctylammonium bromide is used to modify the electrode to induce the natural dye luminophore to produce electrochemiluminescence (ECL); the natural dye luminophore is phycocyanin; and the aqueous system buffer electrolyte is a potassium chloride HEPES buffer electrolyte.

4. The method for constructing an aqueous electrochemiluminescence system based on natural dyes according to claim 3, characterized in that: The aqueous system buffer electrolyte concentration is 1-50 mM and the corresponding pH range is 6-9, the electrolyte concentration is 0-0.3 M; the natural dye luminophore concentration is 0-3 mM; and the tetraoctylammonium bromide concentration is 0-25 mM.

5. A method for detecting the concentration of a substance in an electrochemiluminescent system based on a natural dye, characterized in that: The following steps are involved: Step 1: constructing an electrochemiluminescence system based on the construction method of claim 1 or 3; wherein a solution of a substance to be tested is added to an electrolyte; Step 2: Use an ECL luminometer to record the luminescence intensity of the natural dye luminophore, and perform numerical fitting to obtain a mathematical model based on the luminescence intensity of the natural dye luminophore corresponding to the solutions of the test substance with different concentrations; the concentration of the test substance can be calculated based on the fitted mathematical model and the luminescence intensity corresponding to the solution of the test substance.

6. The method for detecting the substance concentration of the electrochemiluminescence system based on natural dyes according to claim 5, characterized in that: The ECL luminometer parameter settings include: ECL photomultiplier tube bias voltage is −1000~−100 V, the amplification level is 3, the scanning rate is 0~0.3 V / s, and the scanning potential is 0.5~−2.5 V.

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