A chemical timer based on electrochemiluminescence, its application and application methods

Abstract

This invention discloses a chemical timer based on electrochemiluminescence (ECL), its application, and application method. The chemical timer includes an electrochemical workstation, a photomultiplier tube, a computer, a three-electrode system, and an electrolytic cell. The electrolytic cell contains an electrolyte containing an ECL luminescent material and a co-reactant. After applying a constant voltage to the reaction system, the luminescent material and co-reactant generate an intermediate, which then undergoes a subsequent reaction to produce a light signal. Adding a free radical quencher preferentially consumes the intermediate generated during the electrochemical process to delay the ECL signal. By changing the concentration of the free radical quencher, the delay time of the ECL signal can be altered, achieving the effect of a chemical timer. Furthermore, the chemical timer effect is only achieved when the reaction rate between the free radical quencher and the intermediate free radicals is greater than the generation rate of free radicals on the electrode surface, and when the amount of free radical quencher is much smaller than the amount of free radicals generated.

Description

Technical Field

[0001] This invention relates to a chemical timer based on electrochemiluminescence, its application and application method, and belongs to the field of chemical timers. Background Technology

[0002] Electrochemiluminescence (ECL) is a unique form of light emission triggered by an electrochemical reaction. It involves applying a voltage to a reaction system, where the luminescent material and co-reactants undergo redox reactions at the electrode surface to generate intermediates. These intermediates further react with each other or with other substances, causing electrons to transition to an excited state. When these electrons return to the ground state, they emit light of a specific wavelength. Electrochemiluminescence offers many advantages, such as high sensitivity, no need for an excitation source, no interference from external light sources, and precise control over the timing and location of emission.

[0003] Currently, a chemiluminescence-based chemiluminescence timer has been developed, which is a chemiluminescence timer constructed based on the interaction between the luminescent agent luminol and hydrogen peroxide. However, this timer requires the addition of catalysts NaHCO3 and Mn. 2+ Experiments have demonstrated that these two catalysts are essential components in the construction of a chemiluminescence timer. Furthermore, to observe significant chemiluminescence, the luminol and hydrogen peroxide need to reach concentrations in the mM range. Since chemiluminescence occurs instantaneously upon mixing of the luminol and hydrogen peroxide, the chemiluminescence timer starts at the moment of mixing, making the start of the reaction uncontrollable and the timer preparation time insufficient. In addition, the reactants in this chemiluminescence-based timer are limited, only applicable to substances capable of chemiluminescence (such as luminol and acridine esters). Based on these problems with chemiluminescence-based chemiluminescence timers, there is an urgent need to develop an electrochemiluminescence-based chemiluminescence timer that can better address these issues. Summary of the Invention

[0004] Objectives of the Invention: The first objective of this invention is to provide an electrochemiluminescence-based chemiluminescence timer that can be used to detect the concentration of free radical quenchers. The second objective is to provide the application of this chemiluminescence timer in detecting the concentration of free radical quenchers. The third objective is to provide a method for detecting the concentration of free radical quenchers using this chemiluminescence timer.

[0005] Technical Solution: The present invention discloses a chemical timer based on electrochemiluminescence, comprising an electrochemical workstation, a photomultiplier tube, a computer, a three-electrode system, and an electrolytic cell. The electrolytic cell contains an electrolyte containing an ECL luminescent material and a co-reactant. The three-electrode system consists of an ITO working electrode, a silver / silver chloride reference electrode, and a platinum wire counter electrode. The ITO working electrode is positioned in the light signal acquisition area of ​​the photomultiplier tube. The silver / silver chloride reference electrode and the platinum wire counter electrode are both inserted into the electrolyte containing the ECL luminescent material and the co-reactant. The three-electrode system is connected to the electrochemical workstation, and the electromultiplier tube is connected to the computer.

[0006] Furthermore, the ECL luminescent agent is the luminol derivative L012 probe, and the co-reactant is H2O2.

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

[0008] The present invention relates to the application of an electrochemiluminescence-based chemiluminescence timer in detecting the concentration of free radical quenchers.

[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 free radical quenchers using the electrochemiluminescence-based chemiluminescence timer described in the present invention, comprising the following steps:

[0011] (1) Constructing a chemical timer: Place the ITO working electrode in the photomultiplier tube's light signal acquisition area, add an electrolyte containing an ECL luminescent material and a co-reactant to the electrolytic cell, insert the silver / silver chloride reference electrode and platinum wire counter electrode into the electrolyte to form a three-electrode system with the ITO working electrode, connect the three electrodes to the chemical workstation, and connect the chemical workstation and the photomultiplier tube to the computer.

[0012] (2) Trigger the electrochemical workstation, call the constant potential method of the electrochemical workstation, apply voltage for a period of time, and record the ECL intensity change curve over time during that period;

[0013] (3) Add free radical quencher solutions of different concentrations to the electrolyte, call the constant potential method of the electrochemical workstation, apply voltage for a period of time, and stop recording when the ECL signal increases to the highest peak, so as to obtain the ECL intensity change curves with time corresponding to different concentrations of free radical quenchers.

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

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

[0016] Furthermore, in step (1), the concentration of the ECL luminescent material 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 undergo pretreatment, including the following steps:

[0020] (AI) ITO glass was cut into small pieces, ultrasonically treated in deionized and anhydrous ethanol water respectively, and then dried.

[0021] (A2) Cut out hollow circular rubber rings from PDMS, ultrasonically treat them in deionized water and anhydrous ethanol respectively, and then dry them;

[0022] (A3) Fix the dried PDMS hollow ring to the center of the ITO glass conductive surface using molten liquid PDMS, exposing the bottom circular area, and heat to solidify.

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

[0024] This invention, by applying a constant voltage to the reaction system, generates an intermediate from the luminescent material and co-reactant, which then proceeds to produce a light signal. The addition of a free radical quencher preferentially consumes the intermediate generated during the electrochemical process, thus delaying the ECL signal. The delay time of the ECL signal can be altered by changing the concentration of the added free radical quencher, achieving the effect of a chemichronizer. During experiments, it was found that the system could not achieve the chemichronizer effect when using PBS or CBS as buffer. Furthermore, the chemichronizer effect is only achieved when the reaction rate between the free radical quencher and the intermediate free radicals is greater than the rate of free radical generation on the electrode surface, and when the amount of free radical quencher is much smaller than the amount of free radicals generated.

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

[0026] This invention achieves delayed emission of the ECL signal by adding a free radical quencher to the ECL system. This quencher preferentially consumes intermediates generated during the redox reaction of the luminescent probe or co-reactant on the electrode surface during the ECL process. The delay time can be altered by changing the concentration of the free radical quencher, thus establishing a chemiluminescence timer. Compared to chemiluminescence, this method offers greater controllability. Chemiluminescence occurs instantly upon solution mixing, while electrochemiluminescence requires an applied voltage, allowing for more preparation time and facilitating ECL signal collection. Furthermore, electrochemiluminescence does not require a catalyst to enhance the light signal, and the photomultiplier tube can capture minute light signals promptly, making it a more sensitive detection method. In addition, the introduction of a chemiluminescence timer provides better stability compared to conventional ECL intensity measurement methods, avoiding interference from batch-to-batch electrode variations. Attached Figure Description

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

[0028] Figure 2 This is a graph showing the change in ECL intensity over time for the L012-H2O2 electrolyte system in Example 2.

[0029] Figure 3 This is a graph showing the effect of the free radical quencher thiourea on the ECL of the L012-H2O2 system in Example 3.

[0030] Figure 4 This is a graph showing the effect of gallic acid, a free radical quencher, on the ECL of the L012-H2O2 system in Example 4.

[0031] Figure 5 This is a graph showing the effect of ascorbic acid, a free radical quencher, on the ECL of the L012-H2O2 system in Example 5.

[0032] Figure 6 This is a graph showing the effect of PBS buffer on ECL in the L012-H2O2 system in Example 6.

[0033] Figure 7 This is a graph showing the effect of buffer CBS on ECL in the L012-H2O2 system in Example 7.

[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 Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0036] Example 1

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

[0038] (a) Cut the ITO glass into 35mm×35mm pieces, sonicate them in deionized water for 15 minutes and in anhydrous ethanol for 15 minutes, and then dry them in an oven at 60℃.

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

[0040] (c) Remove the solidified PDMS from the culture dish and use a belt to cut out hollow circular rubber rings with an inner diameter of 16 mm and an outer diameter of 22 mm. Sonicate them in deionized water for 15 minutes and in anhydrous ethanol for 15 minutes respectively, and then dry them in a 60°C oven.

[0041] (d) Fix the dried PDMS ring onto the center of the conductive surface of the ITO glass using the unsolidified liquid PDMS mixed in step (b), exposing a circular area with a bottom diameter of 16 mm. Place it on a heating plate at 60°C and allow it to solidify to obtain the ITO working electrode.

[0042] (2) Construction of chemical timer system

[0043] (a) Place the ITO working electrode obtained in step (1) in the photomultiplier tube light signal acquisition area and connect the wire to the working electrode with conductive tape.

[0044] (b) Add 1 mL of an aqueous solution containing 500 μM luminol derivative L012 probe and 1 mM H2O2 to the electrolytic cell as the electrolyte. Insert the silver / silver chloride reference electrode and platinum wire counter electrode into the electrolyte as well. Connect the three leads of the electrochemical workstation to the corresponding three electrodes to complete the chemical timer system setup. The apparatus is as follows: Figure 1 As shown; the device is as follows Figure 1 As shown.

[0045] Example 2: ECL intensity variation curve of the L012-H2O2 system over time

[0046] Using the chemical timer assembled in Example 2, the electrochemical workstation was triggered, and the constant voltage method of the electrochemical workstation was invoked. The constant voltage was set to 0.7V, and the sensitivity was set to 10. -4A. The photomultiplier tube voltage is set to 400V; the electrochemical workstation applies voltage for 600s. During this process, the light emitted from the ITO working electrode surface is collected by the photomultiplier tube, which then transmits the signal to the computer. Simultaneously, the ECL intensity change curve over time is recorded. The results are as follows: Figure 2 .

[0047] Example 3: Effect of the free radical quencher thiourea on the ECL of the L012-H2O2 system (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, respectively, simultaneously triggering the constant voltage method of the electrochemical workstation. The constant voltage was set to 0.7 V, and the sensitivity was 10. -4 A. The photomultiplier tube voltage was set to 400V. Recording was stopped when the ECL signal increased to its peak. Curves showing the change of ECL intensity over time at different concentrations of thiourea were obtained. The logarithm of the added thiourea concentration and the logarithm of the time when the ECL showed an upward trend exhibited a linear relationship between 0.5mM and 40mM. The results are as follows: Figure 3 As shown, Figure 3 The graph shows the effect of the free radical quencher thiourea on the ECL of the L012-H2O2 system in Example 3. In this graph, A represents the change in ECL intensity over time at different thiourea concentrations, and B represents 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 has a linear relationship with the logarithm of the chemical timer delay time, as shown by the equation log(t) = 0.23 × log(c) + 1.59, where t represents the thiourea reaction time (s), c represents the thiourea concentration (mM) after reaction time t, and R... 2 It is 0.9786.

[0049] Example 4: Effect of gallic acid, a free radical quencher, on the ECL of the L012-H2O2 system (curve)

[0050] Using the chemical timer assembled in Example 2, 10 μL of 1 mM, 10 mM, 20 mM, 30 mM, and 40 mM gallic acid, respectively, was added to the electrolyte. Simultaneously, the electrochemical workstation's potentiostatic method was triggered, with the constant voltage set to 0.7 V and the sensitivity set to 10. -4 A. The photomultiplier tube voltage was set to 400V. Recording was stopped when the ECL signal increased to its peak. Curves showing the change of ECL intensity over time at different concentrations of gallic acid were obtained. Furthermore, the logarithm of the added gallic acid concentration and the logarithm of the time it took for the ECL to show an upward trend exhibited a linear relationship between 1mM and 20mM. The results are as follows: Figure 4 As shown, Figure 4This is a graph showing the effect of gallic acid, the free radical quencher, on the ECL of the L012-H2O2 system in Example 4. In Figure A, the ECL intensity changes with time at different gallic acid concentrations, and in Figure B, 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 has a linear relationship with the logarithm of the chemical timer delay time, and 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 concentration of gallic acid after reaction time t (mM), and R... 2 It is 0.9857.

[0051] Example 5: Effect of free radical quencher ascorbic acid on ECL of L012-H2O2 system (curve)

[0052] Using the chemical timer assembled in Example 2, 10 μL of 1 mM, 10 mM, 20 mM, 30 mM, and 40 mM ascorbic acid, a free radical quencher, were added to the electrolyte, respectively. Simultaneously, the constant voltage method of the electrochemical workstation was triggered, with the constant voltage set to 0.7 V and the sensitivity to 10. -4 A. The photomultiplier tube voltage was set to 400V. Recording was stopped when the ECL signal increased to its peak. Curves showing the change of ECL intensity over time at different concentrations of ascorbic acid were obtained. Furthermore, the logarithm of the added ascorbic acid concentration and the logarithm of the time it took for the ECL to show an upward trend exhibited a linear relationship between 1mM and 20mM. The results are as follows: Figure 5 As shown. Figure 5 This is a graph showing the effect of ascorbic acid, a free radical quencher, on the ECL of the L012-H2O2 system in Example 5. In Figure A, the ECL intensity changes over time at different ascorbic acid concentrations, and in Figure B, the logarithm of the ascorbic acid concentration versus the logarithm of time is shown. Figure 5 It can be seen that the logarithm of the added ascorbic acid concentration has a linear relationship with 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 (mM) after reaction time t, and R 2 It is 0.9998.

[0053] Example 6: Effect of PBS buffer on ECL in L012-H2O2 system (curve)

[0054] Using the chemical timer assembled in Example 2, PBS buffer was added to the original electrolyte so that 1 mL of electrolyte contained 500 μM L012 and 1 mM H2O. 2、10 mM PBS was used to add 10 μL of different concentrations of the 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. Simultaneously, the potentiostatic method of the electrochemical workstation was triggered, with the constant voltage set to 0.7 V and the sensitivity set to 10. -4 A. The photomultiplier tube voltage was set to 400V. Recording was stopped when the ECL signal increased to its peak. Curves showing the change in ECL intensity over time for electrolytes with different concentrations of free radical quenchers in PBS buffer were obtained. The results are as follows: Figure 6 As shown. Figure 6 The graphs show the effect of PBS buffer on the ECL of the L012-H2O2 system in Example 6. In Example 6, A shows the ECL intensity over time when thiourea was used as the quencher; B shows the ECL intensity over time when gallic acid was used as the quencher; and C shows the ECL intensity over time when ascorbic acid was used as the quencher. Figure 6 It is evident that the chemichronizer cannot be triggered when the buffer solution is PBS.

[0055] Example 7: Effect of buffer CBS on ECL in L012-H2O2 system (curve)

[0056] Using the chemical timer assembled in Example 2, CBS buffer was added to the original electrolyte so that 1 mL of electrolyte contained 500 μM L012 and 1 mM H2O. 2、 10 mM CBS was used to add 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). Simultaneously, the electrochemical workstation's potentiostatic method was triggered, with a constant voltage set to 0.7 V and a sensitivity of 10. -4 A. The photomultiplier tube voltage was set to 400V. Recording was stopped when the ECL signal increased to its peak. The curves of ECL intensity versus time corresponding to electrolytes with different concentrations of free radical quenchers in CBS buffer were obtained. The results are as follows: Figure 7 As shown. Figure 7 The graphs show the effect of CBS buffer on the ECL of the L012-H2O2 system in Example 7. In Example 7, A shows the ECL intensity over time when thiourea was used as the quencher; B shows the ECL intensity over time when gallic acid was used as the quencher; and C shows the ECL intensity over time when ascorbic acid was used as the quencher. Figure 7It is evident that the chemichronizer cannot be triggered when the buffer solution is CBS.

[0057] Example 8 demonstrates the use of a chemical timer to test the concentration of gallic acid in an 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, respectively. Simultaneously, the constant potential method of the electrochemical workstation was triggered, with the constant voltage set to 0.7 V and the sensitivity to 10. -4 A. The photomultiplier tube voltage was set to 400V. Recording was stopped when the ECL signal increased to its peak, obtaining the ECL intensity versus time curve at that gallic acid concentration. The standard concentrations of the two samples under this chemical timer were obtained based on the standard curve in Example 4. The recovery rates of the two samples were obtained using the formula: Recovery rate = (Standard concentration / Actual concentration) × 100%. The results are as follows: Figure 8 As shown, Figure 8 This is a graph showing the effect of gallic acid, the free radical quencher, on the ECL of the L012-H2O2 system in Example 8. In Example 8, A represents the change in ECL intensity over time at different gallic acid concentrations, and B represents the recovery rates of gallic acid in the actual tap water sample calculated based on the standard curve of Example 4, which are 107.8% and 100.48%, respectively. Figure 8 It is evident that this chemical timer exhibits high recovery rate and good accuracy in detecting free radical quenchers doped in actual water samples, and can be used for the detection of actual samples.

Claims

1. The application of a chemiluminescence-based chemiluminescence timer in detecting the concentration of free radical quenchers, characterized in that, The chemical timer includes an electrochemical workstation, a photomultiplier tube, a computer, a three-electrode system, and an electrolytic cell. The electrolytic cell contains an electrolyte containing an ECL luminescent material and a co-reactant. The three-electrode system consists of an ITO working electrode, a silver / silver chloride reference electrode, and a platinum wire counter electrode. The ITO working electrode is positioned directly in the photomultiplier tube's light signal acquisition area. The silver / silver chloride reference electrode and the platinum wire counter electrode are both immersed in the electrolyte containing the ECL luminescent material and the co-reactant. The three-electrode system is connected to the electrochemical workstation, and the electrochemical workstation and the photomultiplier tube are connected to the computer. The electrolyte does not contain PBS buffer. Alternatively, CBS buffer solution is used to trigger an electrochemical workstation. The workstation's potentiostatic method is invoked, and a voltage is applied for a period of time. The luminescent material and co-reactant generate intermediates, which then proceed to produce a light signal. After adding a free radical quencher, the intermediates generated during the electrochemical process are preferentially consumed, thus delaying the ECL signal. The delay time of the ECL signal is altered by changing the concentration of the added free radical quencher. Quantification is linearly based on the logarithm of the ECL signal delay time versus the logarithm of the free radical quencher concentration. The ECL luminescent material is a luminol derivative L012 probe, and the co-reactant is H2O. 2， The free radical quenchers are thiourea, gallic acid, or ascorbic acid.

2. The application according to claim 1, characterized in that, The electrolyte is an aqueous solution containing an ECL luminescent material and a co-reactant.

3. A method for the application described in claim 1 or 2, characterized in that, Includes the following steps: (1) Constructing a chemical timer: Place the ITO working electrode in the photomultiplier tube light signal acquisition area, add an electrolyte containing ECL luminescent material and co-reactant to the electrolytic cell, insert the silver / silver chloride reference electrode and platinum wire counter electrode into the electrolyte to form a three-electrode system with the ITO working electrode, connect the three electrodes to the electrochemical workstation, and connect the electrochemical workstation and the photomultiplier tube to the computer. (2) Trigger the electrochemical workstation, call the constant potential method of the electrochemical workstation, apply voltage for a period of time, and record the ECL intensity change curve over time during that period; (3) Add different concentrations of free radical quencher solutions to the electrolyte, call the constant potential method of the electrochemical workstation, apply voltage for a period of time, and stop recording when the ECL signal increases to the highest peak, so as to obtain the ECL intensity change curves with time corresponding to different concentrations of free radical quenchers. (4) Add the free radical quencher solution to the electrolyte, call the constant potential method of the electrochemical workstation, apply voltage for a period of time, and stop recording when the ECL signal increases to the highest peak. Use the curve obtained in step (3) to obtain the concentration of the free radical quencher, and use the logarithm of the ECL signal delay time – the logarithm of the concentration of the free radical quencher for linear quantification.

4. The method according to claim 3, characterized in that, In step (1), the concentration of ECL luminescent material in the electrolyte is above 0.5 mM, and the concentration of co-reactant in the electrolyte is above 1 mM.

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

6. The method according to claim 3, characterized in that, In step (1), the ITO working electrode needs to undergo pretreatment, including the following steps: (A1) Cut the ITO glass into small pieces, sonicate them in deionized water and anhydrous ethanol water respectively, and then dry them; (A2) Cut out hollow circular rubber rings from PDMS, sonicate them in deionized water and anhydrous ethanol respectively, and then dry them; (A3) Fix the dried PDMS hollow ring to the center of the ITO glass conductive surface using molten liquid PDMS, exposing the bottom circular area, and heat to solidify.

7. The method according to claim 6, characterized in that, The ultrasonic treatment time is more than 5 minutes, and the heating temperature is 50℃-80℃.

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