Electrochemical timer based on electrochemical luminescence and application and application method thereof

Abstract

The invention discloses a chemical timer based on electrochemical luminescence and application and an application method thereof, the chemical timer comprises an electrochemical workstation, a photomultiplier, a computer, a three-electrode system and an electrolytic tank, and the electrolytic tank is filled with an electrolyte containing an ECL illuminant and a co-reactant. After constant voltage is applied to the reaction system, the luminous body and the co-reactant generate an intermediate, and a subsequent reaction is carried out to generate an optical signal. After the free radical quencher is added, the free radical quencher preferentially consumes an intermediate generated in the electrochemical process to realize delay of an ECL signal, and the delay time of the ECL signal is changed by changing the concentration of the free radical quencher, so that the effect of a chemical timer is achieved. And only when the reaction rate of the free radical quencher and the intermediate free radical is greater than the generation rate of the free radical on the surface of the electrode, and the amount of substance of the free radical quencher is far less than the amount of the free radical generation substance, the effect of a chemical timer can be achieved.

Description

Technical Field

[0001] The invention relates to a chemical timer based on electrochemiluminescence and an application and an application method thereof, belonging to the field of chemical timers. Background Art

[0002] Electrochemiluminescence (ECL) is a unique form of luminescence triggered by an electrochemical reaction. It refers to the application of a certain voltage to the reaction system. The luminophore and co-reactant in the reaction system undergo a redox reaction on the electrode surface to generate intermediates. The intermediates further react with each other or with other substances to cause the electrons to transition to an excited state. When the electrons return to the ground state, they emit light of a certain wavelength. Electrochemiluminescence has many advantages, such as high sensitivity, no need for an excitation light source, no interference from external light sources, and the ability to precisely control the time and position of luminescence.

[0003] Nowadays, a chemical timer based on chemiluminescence has been developed. It is a chemical timer based on the chemiluminescence between the luminophore luminol and hydrogen peroxide, but this timer requires the addition of the catalyst NaHCO 3 With Mn 2+ , experiments have proved that these two catalysts are one of the necessary substances for the construction of chemical timers. And if obvious chemiluminescence is to be observed, the concentrations of the luminophores luminol and hydrogen peroxide need to reach the mM level. Since chemiluminescence occurs at the moment when the luminophores luminol and hydrogen peroxide are mixed, it means that the start time of the chemical timer begins at the moment when the two substances are mixed. Therefore, the start time of the reaction lacks controllability, and the timer preparation time is not sufficient. In addition, the reactants of the chemical timer have limitations and are only applicable to substances that can produce chemiluminescence (such as luminol and acridinium esters). According to the above problems with chemical timers based on chemiluminescence, it is urgent to develop a chemical timer based on electrochemiluminescence that can better solve the above problems. Summary of the invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide a chemical timer based on electrochemiluminescence that can be used to detect the concentration of a free radical quencher. The second purpose of the present invention is to provide the application of the chemical timer in detecting the concentration of a free radical quencher. The third purpose of the present invention is to provide a method for detecting the concentration of a free radical quencher using the chemical timer.

[0005] Technical solution: The chemical timer based on electrochemical luminescence described in the present invention includes an electrochemical workstation, a photomultiplier tube, a computer, a three-electrode system and an electrolytic cell, wherein the electrolytic cell is filled with an electrolyte containing an ECL luminophore and a co-reactant; wherein the three-electrode system is an ITO working electrode, a silver / silver chloride reference electrode and a platinum wire counter electrode, wherein the ITO working electrode is directly placed in the photomultiplier tube light signal collection area, the silver / silver chloride reference electrode and the platinum wire counter electrode are both inserted into the electrolyte containing the ECL luminophore and the co-reactant, the three-electrode system is respectively connected to the electrochemical workstation, and the chemical workstation and the electromultiplier tube are respectively connected to the computer.

[0006] Furthermore, the ECL luminophore is the luminol derivative L012 probe, and the co-reactant is H 2 O 2 .

[0007] Furthermore, the electrolyte is an aqueous solution containing an ECL luminophore and a co-reactant.

[0008] The invention discloses an application of the electrochemiluminescence-based chemical timer in detecting the concentration of a free radical quencher.

[0009] Furthermore, the free radical quencher is thiourea, gallic acid or ascorbic acid.

[0010] The present invention also includes a method for detecting the concentration of a free radical quencher using the electrochemiluminescence-based chemical timer of the present invention, comprising the following steps:

[0011] (1) Constructing a chemical timer: placing an ITO working electrode upright in the light signal collection area of ​​a photomultiplier tube, adding an electrolyte containing an ECL luminophore and a co-reactant into an electrolytic cell, inserting a silver / silver chloride reference electrode and a platinum wire counter electrode into the electrolyte to form a three-electrode system with the ITO working electrode, connecting the three electrodes to a chemical workstation, and connecting the chemical workstation and the photomultiplier tube to a computer;

[0012] (2) triggering the electrochemical workstation, calling the constant potential method of the electrochemical workstation, applying voltage for a period of time, and recording the curve of ECL intensity changing with time during this period;

[0013] (3) adding free radical quencher solutions of different concentrations to the electrolyte, calling the constant potential method of the electrochemical workstation, applying voltage for a period of time, and stopping recording when the ECL signal increases to a peak, thereby obtaining the ECL intensity change curve corresponding to different concentrations of free radical quencher;

[0014] (4) Add the free radical quencher solution to be tested into the electrolyte, call the constant potential method of the electrochemical workstation, apply voltage for a period of time, stop recording when the ECL signal increases to a peak, and use the curve obtained in step (3) to obtain the concentration of the free radical quencher.

[0015] Furthermore, in step (1), the electrolyte cannot contain PBS buffer or CBS buffer.

[0016] Furthermore, in step (1), the concentration of the ECL luminophore in the electrolyte is above 0.5 mM, and the concentration of the co-reactant in the electrolyte is above 1 mM.

[0017] Furthermore, in step (2), the applied voltage is 0.7V-1.5V, and the applied voltage time is 50s-1000s.

[0018] Furthermore, in step (3), the concentration of the free radical quencher solution is 0.5-40 mM.

[0019] 9 Further, in step (1), the ITO working electrode needs to be pre-treated, including the following steps:

[0020] (AI) ITO glass was cut into small pieces, treated with ultrasound in deionized water and anhydrous ethanol for 1 second, and then dried;

[0021] (A2) Cutting the PDMS into hollow circular rubber rings, ultrasonically treating them in deionized water and anhydrous ethanol, respectively, and drying them;

[0022] (A3) The dried PDMS hollow ring rubber ring is fixed to the center of the ITO glass conductive surface using melted liquid PDMS, exposing the bottom circular area, and heated and solidified.

[0023] Furthermore, the ultrasonic treatment time is more than 5 minutes, and the heating temperature is 50°C-80°C.

[0024] After a constant voltage is applied to the reaction system, the luminophore and the co-reactant generate intermediates, thereby performing subsequent reactions to generate light signals. After the free radical quencher is added, the free radical quencher preferentially consumes the intermediates generated in the electrochemical process to achieve the delay of the ECL signal. By changing the concentration of the added free radical quencher, the delay time of the ECL signal is changed to achieve the effect of a chemical timer. During the experiment, it was found that when PBS or CBS was used as a buffer, the system could not achieve the effect of a chemical timer. And only when the reaction rate of the free radical quencher with the intermediate free radical is greater than the generation rate of free radicals on the electrode surface, and the amount of the free radical quencher is much smaller than the amount of the free radical generating substance, will it have the effect of a chemical timer.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0026] The present invention adds a free radical quencher to the ECL system. The addition of the free radical quencher can preferentially consume the intermediates generated after the luminescent probe or the co-reactant undergoes a redox reaction on the electrode surface during the ECL process, thereby achieving the effect of delayed emission of the ECL signal. The delay time is changed by changing the concentration of the free radical quencher, thereby establishing a chemical timer. Compared with chemiluminescence, it is more controllable. Chemiluminescence reacts at the moment of solution mixing, while electrochemical luminescence can only occur after voltage is applied, so there is more preparation time, which is convenient for collecting ECL signals. In addition, no catalyst needs to be added to the electrolyte of electrochemical luminescence to enhance the light signal, and the photomultiplier tube can capture tiny light signals in time, so it is a more sensitive detection method. In addition, compared with the ordinary ECL intensity measurement method, the introduction of the chemical timer also has better stability and can avoid interference from electrode batch differences. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic diagram of the device of the chemical timer in Example 1;

[0028] Figure 2 The electrolyte in Example 2 is L012-H 2 O 2 The ECL intensity of the system changes with time;

[0029] Figure 3 The free radical quencher thiourea in Example 3 is L012-H 2 O 2 System ECL impact change curve;

[0030] Figure 4 The free radical quencher gallic acid in Example 4 is used to treat L012-H 2 O 2 System ECL impact change curve;

[0031] Figure 5 The free radical quencher ascorbic acid in Example 5 is used to treat L012-H 2 O 2 System ECL impact change curve;

[0032] Figure 6 The buffer PBS in Example 6 is used for L012-H 2 O 2 System ECL impact change curve;

[0033] Figure 7 The buffer CBS in Example 7 is used to treat L012-H2 O 2 System ECL impact change curve;

[0034] Figure 8 This is a graph showing the concentration of gallic acid in an actual water sample tested using a chemical timer in Example 8. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0036] Example 1

[0037] (1) Pretreatment of conductive glass ITO working electrode

[0038] (a) The ITO glass was cut into small pieces of 35 mm × 35 mm, ultrasonically treated in deionized water for 15 min and in anhydrous ethanol for 15 min, respectively, and then placed in an oven at 60 °C for drying.

[0039] (b) Add 16-18 g of polydimethylsiloxane (PDMS) component B and 1 / 10 of the mass of component A (about 1.6-1.8 g) to the culture dish, stir in the same direction with a glass rod for 5 minutes to mix them evenly, shake out the bubbles during the mixing process, and then place it on a heating plate at 60°C to wait for it to solidify.

[0040] (c) The solidified PDMS was taken out from the culture dish, and a hollow circular rubber ring with an inner diameter of 16 mm and an outer diameter of 22 mm was punched out using a belt. The ring was ultrasonicated in deionized water for 15 minutes and in anhydrous ethanol for 15 minutes, respectively, and then placed in a 60°C oven for drying.

[0041] (d) The dried PDMS rubber ring was fixed to the center of the ITO glass conductive surface using the unsolidified liquid PDMS mixed in step (b), exposing a circular area with a bottom diameter of 16 mm. It was placed on a heating plate at 60°C and allowed to solidify to obtain an ITO working electrode.

[0042] (2) Construction of chemical timer system

[0043] (a) placing the ITO working electrode obtained in step (1) directly on the photomultiplier tube light signal collection area, and connecting the wire to the working electrode with conductive tape;

[0044] (b) Add 1 mL of 500 μM luminol derivative L012 probe, 1 mM H 2 O 2The aqueous solution is used as the electrolyte, the silver / silver chloride reference electrode and the platinum wire counter electrode are also inserted into the electrolyte, and the three leads of the electrochemical workstation are respectively clamped to the corresponding three electrodes to complete the construction of the chemical timer system. The device is as follows: Figure 1 As shown; the device is as Figure 1 shown.

[0045] Example 2L012-H 2 O 2 Curve of the ECL intensity of the system changing with time

[0046] The chemical timer assembled in Example 2 was used to trigger the electrochemical workstation, and the constant potential method of the electrochemical workstation was called, and the constant voltage was set to 0.7 V and the sensitivity was set to 10 -4 A, the photomultiplier tube high voltage is set to 400V; the electrochemical workstation applies voltage for 600s. During this process, the light emitted from the surface of the ITO working electrode is collected by the photomultiplier tube, which then transmits the signal to the computer and records the ECL intensity change curve over time during this time. The results are shown in the figure. Figure 2 .

[0047] Example 3 Free Radical Quencher Thiourea to L012-H 2 O 2 System ECL impact change curve

[0048] Using the chemical timer assembled in Example 2, 10 μL of 0.5 mM, 1 mM, 10 mM, 20 mM, 30 mM and 40 mM free radical quencher thiourea were added to the electrolyte, and the constant potential method of the electrochemical workstation was triggered at the same time, and the constant voltage was set to 0.7 V and the sensitivity was set to 10 -4 A, the photomultiplier tube high voltage was set to 400V, and the recording was stopped when the ECL signal increased to the highest peak. The ECL intensity change curve with time under different thiourea concentrations was obtained, and the logarithm of the added thiourea concentration and the logarithm of the time when the ECL showed an upward trend showed a linear change between 0.5mM and 40mM. The results are shown in Figure 3 As shown, Figure 3 The free radical quencher thiourea in Example 3 is L012-H 2 O 2 The ECL effect change curve of the system is shown in Figure 1, where A is the change curve of ECL intensity over time at different thiourea concentrations, and B is the linear relationship between the logarithm of thiourea concentration and the logarithm of time. Figure 3 It can be seen that the logarithm of the added thiourea concentration and the logarithm of the chemical timer delay time are linearly related. The equation is log(t)=0.23×log(c)+1.59, where t represents the thiourea reaction time (s), c represents the thiourea concentration after the reaction time t (mM), and R 2 It is 0.9786.

[0049] Example 4 Free Radical Quencher Gallic Acid to L012-H 2 O 2 System ECL impact change curve

[0050] Using the chemical timer assembled in Example 2, 10 μL of 1 mM, 10 mM, 20 mM, 30 mM and 40 mM free radical quencher gallic acid were added to the electrolyte, and the constant potential method of the electrochemical workstation was triggered at the same time, and the constant voltage was set to 0.7 V and the sensitivity was set to 10 -4 A, the photomultiplier tube high voltage was set to 400V, and the recording was stopped when the ECL signal increased to the highest peak. The ECL intensity change curve with time under different concentrations of gallic acid was obtained, and the logarithm of the added gallic acid concentration and the logarithm of the time when the ECL showed an upward trend showed a linear change between 1mM and 20mM. The results are shown in Figure 4 As shown, Figure 4 The free radical quencher gallic acid in Example 4 is used to treat L012-H 2 O 2 The ECL effect change curve of the system, where A is the change curve of ECL intensity over time at different gallic acid concentrations, and B is the linear relationship between the logarithm of gallic acid concentration and the logarithm of time. Figure 4 It can be seen that the logarithm of the added gallic acid concentration and the logarithm of the chemical timer delay time are linearly related. The equation is log(t / s)=0.56×log(c / mM)+1.49, where t represents the gallic acid reaction time (s), c represents the gallic acid concentration after the reaction time t (mM), and R 2 It is 0.9857.

[0051] Example 5: Effect of free radical quencher ascorbic acid on L012-H 2 O 2 System ECL impact change curve

[0052] Using the chemical timer assembled in Example 2, 10 μL of 1 mM, 10 mM, 20 mM, 30 mM and 40 mM free radical quencher ascorbic acid were added to the electrolyte, respectively, and the constant potential method of the electrochemical workstation was triggered at the same time, and the constant voltage was set to 0.7 V and the sensitivity was set to 10 -4 A, the photomultiplier tube high voltage was set to 400V, and the recording was stopped when the ECL signal increased to the peak. The ECL intensity change curve with time under different ascorbic acid concentrations was obtained, and the logarithm of the added ascorbic acid concentration and the logarithm of the time when the ECL showed an upward trend showed a linear change between 1mM and 20mM. The results are shown in Figure 5 shown. Figure 5 The free radical quencher ascorbic acid in Example 5 is used to treat L012-H2 O 2 The ECL effect change curve of the system, where A is the change curve of ECL intensity over time at different ascorbic acid concentrations, and B is the linear relationship between the logarithm of ascorbic acid concentration and the logarithm of time. Figure 5 It can be seen that the logarithm of the added ascorbic acid concentration is linearly related to the logarithm of the chemical timer delay time, and the equation is log(t / s)=0.71×log(c / mM)+1.34, where t represents the ascorbic acid reaction time (s), c represents the ascorbic acid concentration after the reaction time t (mM), and R 2 is 0.9998.

[0053] Example 6 PBS buffer for L012-H 2 O 2 System ECL impact change curve

[0054] Using the chemical timer assembled in Example 2, PBS buffer was added to the original electrolyte to make 1 mL of the electrolyte contain 500 μM L012, 1 mM H 2 O 2、 10 mM PBS was added with 10 μL of different concentrations of free radical quenchers thiourea (0.5 mM, 1 mM, 10 mM, 20 mM, 30 mM and 40 mM), gallic acid (1 mM, 10 mM, 20 mM, 30 mM and 40 mM), and ascorbic acid (1 mM, 10 mM, 20 mM, 30 mM and 40 mM), respectively. The constant potential method of the electrochemical workstation was triggered at the same time, and the constant voltage was set to 0.7 V and the sensitivity was set to 10 -4 A, the photomultiplier tube high voltage is set to 400V, and the recording is stopped when the ECL signal increases to the highest peak. The ECL intensity curve corresponding to the electrolyte with different concentrations of free radical quencher in the buffer solution of PBS is obtained. The results are shown in Figure 6 shown. Figure 6 The buffer PBS in Example 6 is used for L012-H 2 O 2 The ECL effect change curve of the system, where A is the change curve of ECL intensity over time when the quencher is thiourea, B is the change curve of ECL intensity over time when the quencher is gallic acid, and C is the change curve of ECL intensity over time when the quencher is ascorbic acid. Figure 6 It can be seen that when the buffer is PBS, the chemical timer cannot be triggered.

[0055] Example 7 Buffer CBS to L012-H 2 O 2 System ECL impact change curve

[0056] Using the chemical timer assembled in Example 2, CBS buffer was added to the original electrolyte to make 1 mL of the electrolyte contain 500 μM L012, 1 mM H 2 O 2、 10 mM CBS, 10 μL of different concentrations of free radical quenchers thiourea (0.5 mM, 1 mM, 10 mM, 20 mM, 30 mM and 40 mM), gallic acid (1 mM, 10 mM, 20 mM, 30 mM and 40 mM), ascorbic acid (1 mM, 10 mM, 20 mM, 30 mM and 40 mM) were added, and the constant potential method of the electrochemical workstation was triggered at the same time, and the constant voltage was set to 0.7 V and the sensitivity was 10 -4 A, the photomultiplier tube high voltage is set to 400V, and the recording is stopped when the ECL signal increases to the highest peak. The ECL intensity curve corresponding to the electrolyte with different concentrations of free radical quencher in the buffer solution CBS is obtained. The results are shown in Figure 7 shown. Figure 7 The buffer CBS in Example 7 is used to treat L012-H 2 O 2 The ECL effect change curve of the system, where A is the change curve of ECL intensity over time when the quencher is thiourea, B is the change curve of ECL intensity over time when the quencher is gallic acid, and C is the change curve of ECL intensity over time when the quencher is ascorbic acid. Figure 7 It can be seen that when the buffer is CBS, the chemical timer cannot be triggered.

[0057] Example 8 is to use a chemical timer to test the gallic acid concentration in the actual water sample

[0058] Using the chemical timer assembled in Example 2, 10 μL of actual tap water samples containing 15 mM and 25 mM gallic acid were added to the electrolytic cell, and the constant potential method of the electrochemical workstation was triggered at the same time, with the constant voltage set to 0.7 V and the sensitivity set to 10 -4 A, the photomultiplier tube high voltage is set to 400V, and the recording is stopped when the ECL signal increases to the highest peak, and the ECL intensity curve with time at this concentration of gallic acid is obtained. The standard concentrations of the two samples under this chemical timer are obtained according to the standard curve of Example 4, and the recovery rates of the two samples are obtained according to the formula: recovery rate = (standard concentration / actual concentration) × 100%. The results are shown in Figure 8 As shown, Figure 8 The free radical quencher gallic acid in Example 8 is used to treat L012-H 2 O 2The ECL effect curve of the system, where A is the curve of the change of ECL intensity over time at different gallic acid concentrations, and B is the recovery rate of gallic acid in the actual tap water sample calculated according to the standard curve in Example 4, which are 107.8% and 100.48% respectively. Figure 8 It can be seen that the chemical timer has a high recovery rate and good accuracy for detecting free radical quenchers doped in actual water samples, and can be used for the detection of actual samples.

Claims

1. A chemical timer based on electrochemiluminescence, characterized in that: It includes an electrochemical workstation, a photomultiplier tube, a computer, a three-electrode system and an electrolytic cell, wherein the electrolytic cell is filled with an electrolyte containing an ECL luminophore and a co-reactant; wherein the three-electrode system is an ITO working electrode, a silver / silver chloride reference electrode and a platinum wire counter electrode, wherein the ITO working electrode is placed directly in the photomultiplier tube optical signal collection area, the silver / silver chloride reference electrode and the platinum wire counter electrode are both inserted into the electrolyte containing the ECL luminophore and the co-reactant, the three-electrode system is respectively connected to the electrochemical workstation, and the chemical workstation and the electromultiplier tube are respectively connected to the computer.

2. The electrochemiluminescence-based chemical timer according to claim 1, characterized in that: The ECL luminophore is the luminol derivative L012 probe, and the co-reactant is H2O2.

3. The electrochemiluminescence-based chemical timer according to claim 1, characterized in that: The electrolyte is an aqueous solution containing an ECL luminophore and a co-reactant.

4. Use of the electrochemiluminescence-based chemical timer according to any one of claims 1 to 3 in detecting the concentration of a free radical quencher.

5. The use according to claim 4, characterized in that The free radical quencher is thiourea, gallic acid or ascorbic acid.

6. A method for detecting the concentration of a free radical quencher using the electrochemiluminescence-based chemical timer according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Constructing a chemical timer: placing an ITO working electrode upright in the light signal collection area of ​​a photomultiplier tube, adding an electrolyte containing an ECL luminophore and a co-reactant into an electrolytic cell, inserting a silver / silver chloride reference electrode and a platinum wire counter electrode into the electrolyte to form a three-electrode system with the ITO working electrode, connecting the three electrodes to a chemical workstation, and connecting the chemical workstation and the photomultiplier tube to a computer; (2) triggering the electrochemical workstation, calling the constant potential method of the electrochemical workstation, applying voltage for a period of time, and recording the curve of the ECL intensity changing with time during this period; (3) adding free radical quencher solutions of different concentrations to the electrolyte, calling the constant potential method of the electrochemical workstation, applying voltage for a period of time, and stopping recording when the ECL signal increases to a peak, thereby obtaining the ECL intensity change curve corresponding to different concentrations of free radical quencher; (4) Add the free radical quencher solution to be tested into the electrolyte, call the constant potential method of the electrochemical workstation, apply voltage for a period of time, stop recording when the ECL signal increases to a peak, and use the curve obtained in step (3) to obtain the concentration of the free radical quencher.

7. The method according to claim 6, characterized in that In step (1), the electrolyte cannot contain PBS buffer or CBS buffer, the concentration of ECL luminophore in the electrolyte is above 0.5 mM, and the concentration of co-reactant in the electrolyte is above 1 mM.

8. The method according to claim 6, characterized in that In step (2), the applied voltage is 0.7V-1.5V, and the applied voltage time is 50s-1000s. In step (3), the concentration of the free radical quencher solution is 0.5-40mM.

9. The method according to claim 6, characterized in that In step (1), the ITO working electrode needs to be pre-treated, including the following steps: (AI) ITO glass was cut into small pieces, treated with ultrasound in deionized water and anhydrous ethanol for 1 second, and then dried; (A2) Cutting the PDMS into hollow circular rubber rings, ultrasonically treating them in deionized water and anhydrous ethanol, respectively, and drying them; (A3) The dried PDMS hollow ring rubber ring is fixed to the center of the ITO glass conductive surface using melted liquid PDMS, exposing the bottom circular area, and heated and solidified.

10. The method according to claim 9, characterized in that The ultrasonic treatment time is more than 5 minutes, and the heating temperature is 50°C-80°C.

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