A pulse voltage test method for suppressing the oxidation of a gold electrode in electrochemical analysis

Abstract

The present invention discloses a pulse voltage test method for inhibiting the oxidation of a gold electrode in electrochemical analysis, comprising the following steps: Step 1, placing a gold electrode or a glassy carbon electrode modified with gold nanoparticles, a counter electrode, and a reference electrode into a solution to be analyzed for electrochemical testing; Step 2, applying a pulsed constant voltage of 0.01 - 1 s and 0.6 - 1.1 V, and an oxidation reaction occurs on the electrode surface to generate an electrochemical signal; Step 3, applying a pulsed constant voltage of 0.3 - 1 s and -0.5 - 0.5 V, and a reduction reaction occurs on the electrode surface to reduce the reversibly generated gold oxide during the oxidation stage; Step 4, repeating Steps 2 - 3 more than 5 times. The present invention uses the pulse voltage method for electrochemical analysis on the surface of the gold electrode, enabling the gold to be immediately reduced after being oxidized at a high potential, ensuring the stability of the electrode properties, and greatly improving the stability of the electrochemical signal; it can be applied to the electrochemiluminescence test of ruthenium tris(bipyridine), broadening the analysis scope.

Description

Technical Field

[0001] The present invention belongs to the voltage testing method, specifically a pulsed voltage testing method for suppressing the oxidation of gold electrodes in electrochemical analysis. Background Art

[0002] Experimental results and literature studies have shown that voltages above 0.6 V (relative to the silver chloride reference electrode) in aqueous solutions will cause reversible oxidation of gold, generating gold oxide Au-O chemisorbed on the electrode surface, and voltages above 1.1 V will cause further irreversible oxidation of gold to form Au 2 O 3 , and these gold oxides seriously hinder electrochemical analysis. Conventional cyclic voltammetry and potentiostatic testing cannot effectively solve this problem, so existing reports often use low-potential analysis on the gold electrode surface or avoid using gold electrodes when higher potentials are required. Pulsed voltage testing is a transient electrochemical testing technique, the main feature of which is to apply periodic and short-term voltage pulses to the electrode, and the voltage change is discontinuous. The advantages of this testing method are high signal quality, flexible pulse adjustment, small impact on sample properties, and low total energy consumption. It is applied to testing scenarios such as multi-substance determination and ion chromatography coupled with electrochemical analysis.

[0003] This testing method can broaden the application scenarios of oxidative electrochemical analysis on the gold electrode surface. For example, electrochemiluminescence (or electrochemically generated chemiluminescence) is widely used in various sensors due to its low background response and high sensitivity. Gold electrodes and electrodes modified with gold nanoparticles are often used to assemble biorecognition elements or immobilize luminophores due to their good biocompatibility and strong interaction with thiols, and are one of the commonly used electrodes in electrochemiluminescence testing. However, most existing electrochemiluminescence tests only consider the redox reaction potential of the luminophore and pay less attention to the reactions occurring on the electrode surface. The commercial tris(2,2'-bipyridyl)ruthenium(III) / tripropylamine system electrochemically oxidizes tris(2,2'-bipyridyl)ruthenium(III) (1.14 V), and its main luminescence potential is at 0.9 V or 1.14 V. The analysis at this potential will be interfered by gold oxidation. Steady-state electrochemical testing methods such as cyclic voltammetry or constant voltage method cannot solve the oxidation problem, resulting in the attenuation of electrochemiluminescence signals and affecting quantitative determination. There is currently no report on applying the pulsed voltage method to electrochemiluminescence analysis on the gold electrode surface. Setting a suitable pulse method for the gold redox behavior is expected to achieve stable high-potential electrochemiluminescence testing on the gold electrode surface. Summary of the Invention

[0004] Object of the Invention: In order to overcome the deficiencies in the prior art, the object of the present invention is to provide two pulsed voltage testing methods for realizing stable and efficient oxidation reactions on the gold electrode surface and suppressing the oxidation of gold electrodes in electrochemical analysis.

[0005] Technical solution: A pulsed voltage test method for inhibiting the oxidation of a gold electrode in electrochemical analysis according to the present invention comprises the following steps:

[0006] Step 1, placing a gold electrode or a glassy carbon electrode modified with gold nanoparticles, a counter electrode, and a reference electrode into a solution to be analyzed, and performing electrochemical testing;

[0007] Step 2, applying a pulsed constant voltage of 0.01 - 1 s and 0.6 - 1.1 V, and an oxidation reaction occurs on the surface of the gold electrode or the glassy carbon electrode modified with gold nanoparticles to generate an electrochemiluminescence signal;

[0008] Step 3, applying a pulsed constant voltage of 0.3 - 1 s and -0.5 - 0.5 V, and a reduction reaction occurs on the surface of the gold electrode or the glassy carbon electrode modified with gold nanoparticles to reduce the reversibly generated gold oxide during the oxidation stage;

[0009] Step 4, repeating Steps 2 - 3 more than 5 times to obtain sufficient data for analyzing the signal quality, avoiding the oxidation effect of the gold electrode or the glassy carbon electrode modified with gold nanoparticles, and obtaining electrochemical or optical signals with higher quality than the non-pulsed method.

[0010] Further, the counter electrode is a platinum wire. The reference electrode is a silver chloride electrode or a saturated calomel electrode. The electrochemical analysis solution is an aqueous solution containing an electrolyte. The solution to be analyzed is a 10-fold phosphate buffer solution of ruthenium (II) tris(2,2'-bipyridyl) dichloride and tripropylamine. The pH of the 10-fold phosphate buffer solution is 7.2 - 7.4.

[0011] Further, after Step 2, a voltage of 0 V is applied for 0.5 - 2 s to return the system to the equilibrium state.

[0012] Further, after Step 3, a voltage of 0 V is applied for 0.5 - 2 s to return the system to the equilibrium state.

[0013] Working principle: The pulsed voltage test method is mainly based on the redox properties of gold, that is, gold is reversibly oxidized to form Au - O at 0.6 - 1.1 V, and this oxide can be reduced at a voltage of <0.5 V; gold is irreversibly oxidized to Au 2 O 3 , and this oxide cannot be reduced. The present invention uses a transient test method - the pulsed voltage method, applying an instantaneous high pulsed voltage (<1.1 V) to the gold electrode (oxidation stage) to generate an electrochemiluminescence signal, and then applying a slightly longer low pulsed voltage (<0.5 V) to reduce the generated Au - O. In addition, each cycle also includes an equilibrium stage (0 V). The instantaneous high pulsed voltage can minimize the oxidation degree of gold and generate high-quality electrochemiluminescence signals, and the low pulsed voltage in the reduction stage effectively reduces the generated gold oxide, returning the gold electrode to the state before oxidation.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following remarkable features:

[0015] 1. Innovatively using the pulsed voltage method for electrochemical oxidation analysis on the surface of the gold electrode, the gold is immediately reduced after being briefly oxidized at a high potential, ensuring the stability of the electrode properties and greatly improving the stability of the electrochemical signal;

[0016] 2. Using the pulse method for testing, where the voltage changes in a stepwise manner rather than at a constant rate, greatly shortening the detection time and improving the detection efficiency;

[0017] 3. Using the pulsed voltage method for electrochemical analysis, the voltage is constant during the oxidation stage, and the reaction efficiency is higher and the signal quality is better compared with the commonly used cyclic voltammetry. Brief description of the drawings

[0018] Figure 1 is the test schematic diagram of the present invention;

[0019] Figure 2 is the current-time graph of the 0.3s pulsed voltage test on the surface of the gold electrode of the present invention;

[0020] Figure 3 is the result of the electrochemiluminescence test with a 0.3s pulsed voltage on the surface of the gold electrode of the present invention;

[0021] Figure 4 is the current-time graph of the 0.5s pulsed voltage test on the surface of the gold electrode of the present invention;

[0022] Figure 5 is the result of the electrochemiluminescence test with a 0.5s pulsed voltage on the surface of the gold electrode of the present invention;

[0023] Figure 6 is the current-time graph of the 1s pulsed voltage test on the surface of the gold electrode of the present invention;

[0024] Figure 7 is the result of the electrochemiluminescence test with a 1s pulsed voltage on the surface of the gold electrode of the present invention;

[0025] Figure 8 is the electrochemiluminescence response graph obtained by cyclic voltammetry scanning of the ruthenium tris(bipyridyl), tripropylamine system on the surface of the gold nanoparticle-modified glassy carbon electrode and the bare glassy carbon electrode of the present invention;

[0026] Figure 9 is the result of the cyclic voltammetry scanning of the gold nanoparticle-modified glassy carbon electrode in the ruthenium tris(bipyridyl) / tripropylamine solution from -0.5 to 1.3V, where A is the light intensity-voltage graph and B is the light intensity-time graph;

[0027] Figure 10This is the result of cyclic voltammetry scanning of the glassy carbon electrode modified with gold nanoparticles of the present invention in a ruthenium tris(bipyridine) / tripropylamine solution at -0.5 to 0.9 V. Among them, A is the light intensity-voltage diagram, and B is the light intensity-time diagram;

[0028] Figure 11 This is the result of cyclic voltammetry scanning of the glassy carbon electrode modified with gold nanoparticles of the present invention in a ruthenium tris(bipyridine) / tripropylamine solution at 0.5 to 0.9 V. Among them, A is the light intensity-voltage diagram, and B is the light intensity-time diagram;

[0029] Figure 12 This is the light intensity-voltage diagram of cyclic voltammetry scanning of the gold electrode sensor immobilized with ruthenium tris(bipyridine) of the present invention at -1.3 to 1.3 V;

[0030] Figure 13 This is the instantaneous pulse voltage analysis method used for the gold electrode sensor immobilized with ruthenium tris(bipyridine);

[0031] Figure 14 This is the electrochemical luminescence response result of the gold electrode sensor immobilized with ruthenium tris(bipyridine) measured by the instantaneous pulse voltage method. Specific embodiments

[0032] In the following examples, the silver / silver chloride reference electrode can be abbreviated as the silver chloride electrode. The gold electrode is purchased from Tianjin Aida Hengsheng Technology Development Co., Ltd., with an inner diameter of 5 mm. The used glassy carbon electrode is purchased from Shanghai Chenhua Instrument Co., Ltd., with an inner diameter of 3 mm. The 10-fold PBS solution is purchased directly, and the pH is 7.2 - 7.4.

[0033] Example 1

[0034] Such as Figure 1 , a pulse voltage test method for inhibiting the oxidation of the gold electrode in electrochemical analysis, including the following steps:

[0035] a. Polish the gold electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm of ɑ-Al 2 O 3 Then, ultrasonically clean it alternately with ethanol and ultrapure water 3 times, 30 seconds each time. In a 10-fold PBS solution containing 1 μM ruthenium tris(bipyridine) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, use the gold electrode as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride as the reference electrode for electrochemical testing.

[0036] b. Oxidation stage: Apply a pulsed constant voltage of 0.3 s and 0.9 V. An oxidation reaction occurs on the surface of the gold electrode, and tripropylamine is oxidized to generate a current signal. The luminescence of ruthenium tris(bipyridine) at 0.9 V in the presence of a high-concentration coreactant tripropylamine is used for testing, avoiding the oxidation of the gold electrode caused by the high potential of 1.14 V in the conventional electrochemical luminescence, and improving the analysis credibility.

[0037] c. Reduction stage: Apply a pulsed constant voltage of 0.3 s and -0.5 V. A reduction reaction occurs on the surface of the gold electrode, which is used to reduce the reversibly generated gold oxide during the oxidation stage.

[0038] d. Repeat steps b - c 30 times to obtain sufficient data for analyzing the signal quality.

[0039] As Figure 2 shown, with the change of the pulsed voltage, the oxidation and reduction reactions on the sensor surface alternate, and the electrochemical workstation records a stable current signal. The results show that this pulsed test method avoids the oxidation effect of the gold electrode and obtains high-quality electrochemical signals.

[0040] Example 2

[0041] A pulsed voltage test method for suppressing the oxidation of gold electrodes in electrochemical analysis includes the following steps:

[0042] a. Polish the gold electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α - Al 2 O 3 and then ultrasonically clean it alternately with ethanol and ultrapure water 3 times, 30 seconds each time. In a 10-fold PBS solution containing 1 μM ruthenium tris(bipyridine) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, use the gold electrode as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride as the reference electrode for electrochemical luminescence testing. Use the Xi'an Ruimai MPI - A type electrochemical luminescence analyzer for analysis, with a photomultiplier tube high voltage of 700 V, a sampling frequency of 10 T / s, an amplification factor of 3, a stationary time of 2 s, and a sensitivity of 1×10 -3 A / V.

[0043] b. Oxidation stage: Apply a pulsed constant voltage of 0.3 s and 0.9 V. An oxidation reaction occurs on the surface of the gold electrode, which is used to generate an electrochemical luminescence signal.

[0044] c. Reduction stage: Apply a pulsed constant voltage of 0.3 s and -0.5 V. A reduction reaction occurs on the surface of the gold electrode, which is used to reduce the reversibly generated gold oxide during the oxidation stage.

[0045] d. Repeat steps b - c 30 times to obtain sufficient data for analyzing the signal quality.

[0046] As Figure 3 shown, when the voltage pulse is 0.9 V, the electrochemiluminescence analyzer records a stable optical signal, and the relative standard deviation RSD is only 1.06%, and the signal is stable. The results show that this pulsed test method avoids the oxidation effect of the gold electrode and obtains high-quality electrochemiluminescence signals.

[0047] Example 3

[0048] A pulsed voltage test method for suppressing the oxidation of gold electrodes in electrochemical analysis includes the following steps:

[0049] a. Polish the gold electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al 2 O 3 and then ultrasonically clean it alternately with ethanol and ultrapure water 3 times, 30 seconds each time. In a 10-fold PBS solution containing 1 μM ruthenium(III) tris(bipyridine) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, use the gold electrode as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode as the reference electrode for electrochemical testing.

[0050] b. Oxidation stage: Apply a pulsed constant voltage of 0.5 s and 0.9 V. An oxidation reaction occurs on the surface of the gold electrode, and tripropylamine is oxidized to generate a current signal.

[0051] c. Reduction stage: Apply a pulsed constant voltage of 0.5 s and -0.5 V. A reduction reaction occurs on the surface of the gold electrode to reduce the reversibly formed gold oxide during the oxidation stage.

[0052] d. Repeat steps b - c 85 times to obtain sufficient data for analyzing the signal quality.

[0053] As Figure 4 shown, as the pulsed voltage changes, the oxidation and reduction currents on the sensor surface alternate. When the voltage pulse is 0.9 V, the electrochemical workstation records a stable current signal. The results show that the test method with a pulsed voltage duration of 0.5 s avoids the oxidation effect of the gold electrode and obtains high-quality electrochemical signals.

[0054] Example 4

[0055] A pulsed voltage test method for suppressing the oxidation of gold electrodes in electrochemical analysis includes the following steps:

[0056] a. Polish the gold electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al 2 O 3The gold electrode was polished and then ultrasonically cleaned 3 times alternately with ethanol and ultrapure water for 30 seconds each time. In a 10-fold PBS solution containing 1 μM ruthenium (II) tris(2,2'-bipyridyl) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, using the gold electrode as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode as the reference electrode, electrochemiluminescence testing was carried out. Analysis was performed using a Xi'an Ruimai MPI-A type electrochemiluminescence analyzer, with the high voltage of the photomultiplier tube being 700 V, the sampling frequency being 10 T / s, the amplification factor being 3, the stationary time being 2 s, and the sensitivity being 1×10 -3 A / V.

[0057] b. Oxidation stage: Apply a pulsed constant voltage of 0.9 V for 0.5 s, and an oxidation reaction occurs on the surface of the gold electrode to generate an electrochemiluminescence signal.

[0058] c. Reduction stage: Apply a pulsed constant voltage of -0.5 V for 0.5 s, and a reduction reaction occurs on the surface of the gold electrode to reduce the gold oxide reversibly generated during the oxidation stage.

[0059] d. Repeat steps b - c 85 times to obtain sufficient data for analyzing the signal quality.

[0060] As Figure 5 shown, when the voltage pulse is 0.9 V, the electrochemiluminescence analyzer records a stable light signal, with the relative standard deviation RSD being only 3.52%, and the signal is stable. The results show that the test method with a pulsed voltage duration of 0.5 s avoids the oxidation effect of the gold electrode and obtains a high-quality electrochemiluminescence signal.

[0061] Example 5

[0062] A pulsed voltage test method for suppressing the oxidation of gold electrodes in electrochemical analysis, comprising the following steps:

[0063] a. Use metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α - Al 2 O 3 in sequence to polish the gold electrode, and then ultrasonically clean it 3 times alternately with ethanol and ultrapure water for 30 seconds each time. In a 10-fold PBS solution containing 1 μM ruthenium (II) tris(2,2'-bipyridyl) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, using the gold electrode as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride electrode as the reference electrode, electrochemiluminescence testing was carried out. Analysis was performed using a Xi'an Ruimai MPI-A type electrochemiluminescence analyzer, with the high voltage of the photomultiplier tube being 700 V, the sampling frequency being 10 T / s, the amplification factor being 3, the stationary time being 2 s, and the sensitivity being 1×10 -3 A / V.

[0064] b. Oxidation stage: Apply a pulsed constant voltage of 1 s and 0.9 V. An oxidation reaction occurs on the surface of the gold electrode, and tripropylamine is oxidized to generate a current signal.

[0065] c. Reduction stage: Apply a pulsed constant voltage of 1 s and -0.5 V. A reduction reaction occurs on the surface of the gold electrode to reduce the reversibly formed gold oxide during the oxidation stage.

[0066] d. Repeat steps b - c 100 times to obtain sufficient data for analyzing the signal quality.

[0067] As Figure 6 shown, as the pulsed voltage changes, the oxidation and reduction currents on the sensor surface alternate, and the electrochemical workstation records a stable signal. The results show that the test method with a pulsed voltage duration of 1 s avoids the oxidation effect of the gold electrode and obtains high-quality electrochemiluminescence signals.

[0068] Example 6

[0069] A pulsed voltage test method for suppressing the oxidation of gold electrodes in electrochemical analysis, comprising the following steps:

[0070] a. Polish the gold electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al 2 O 3 and then ultrasonically clean it alternately with ethanol and ultrapure water 3 times, 30 seconds each time. In a 10-fold PBS solution containing 1 μM ruthenium (II) tris(bipyridine) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, use the gold electrode as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride electrode as the reference electrode for electrochemiluminescence testing. Analyze using a Xi'an Ruimai MPI-A type electrochemiluminescence analyzer, with a photomultiplier tube high voltage of 700 V, a sampling frequency of 10 T / s, an amplification factor of 3, a dwell time of 2 s, and a sensitivity of 1×10 -3 A / V.

[0071] b. Oxidation stage: Apply a pulsed constant voltage of 1 s and 0.9 V. An oxidation reaction occurs on the surface of the gold electrode to generate an electrochemiluminescence signal.

[0072] c. Reduction stage: Apply a pulsed constant voltage of 1 s and -0.5 V. A reduction reaction occurs on the surface of the gold electrode to reduce the reversibly formed gold oxide during the oxidation stage.

[0073] d. Repeat steps b - c 100 times to obtain sufficient data for analyzing the signal quality.

[0074] As Figure 7As shown, with the change of the pulsed voltage, when the voltage pulse is 0.9 V, the electrochemiluminescence analyzer records a stable optical signal, and the relative standard deviation RSD is only 3.95%, and the signal is stable. The results show that the test method with a pulsed voltage duration of 1 s avoids the oxidation effect of the gold electrode and obtains high-quality electrochemiluminescence signals.

[0075] Example 7

[0076] A pulsed voltage test method for suppressing the oxidation of gold electrodes in electrochemical analysis, comprising the following steps:

[0077] a. Polish the glassy carbon electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al 2 O 3 and then ultrasonically clean it alternately with ethanol and ultrapure water 3 times, 30 seconds each time. Finally, perform CV scanning in a 0.5 mM sulfuric acid solution until stable. Insert the polished glassy carbon electrode into a 10-fold PBS buffer solution of 2 mM chloroauric acid, use the glassy carbon electrode as the working electrode, silver / silver chloride electrode as the reference electrode, and platinum wire as the counter electrode, and perform gold deposition on the surface of the glassy carbon electrode at a constant potential of -0.2 V. Control the deposition charge to be 1 mC to obtain a glassy carbon electrode modified with gold nanoparticles (AuNPs@GCE). In a 10-fold PBS solution containing 1 μM ruthenium (II) tris(2,2'-bipyridyl) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, use the glassy carbon electrode modified with gold nanoparticles as the working electrode, platinum wire as the counter electrode, and saturated calomel electrode as the reference electrode to perform electrochemiluminescence testing.

[0078] b. Oxidation stage: Apply a pulsed constant voltage of 0.01 s and 1.1 V, and an oxidation reaction occurs on the surface of the glassy carbon electrode modified with gold nanoparticles to generate electrochemiluminescence signals.

[0079] c. Reduction stage: Apply a pulsed constant voltage of 0.3 s and 0.5 V, and a reduction reaction occurs on the surface of the glassy carbon electrode modified with gold nanoparticles to reduce the reversibly generated gold oxide in the oxidation stage.

[0080] d. Repeat steps b - c 5 times to obtain sufficient data for analyzing the signal quality.

[0081] Example 8

[0082] Comparison of electrochemiluminescence signals obtained by the pulsed voltage method and cyclic voltammetry:

[0083] Polish the glassy carbon electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al 2 O 3The gold electrode was polished and then ultrasonically cleaned alternately with ethanol and ultrapure water three times for 30 seconds each time. Finally, CV scanning was performed in a 0.5 mM sulfuric acid solution until stable. The polished glassy carbon electrode was inserted into a 10-fold PBS buffer solution of 2 mM chloroauric acid. Using the glassy carbon electrode as the working electrode, silver / silver chloride electrode as the reference electrode, and platinum wire as the counter electrode, gold deposition was carried out on the surface of the glassy carbon electrode at a constant potential of -0.2 V. The deposition charge was controlled to be 1 mC to obtain a glassy carbon electrode modified with gold nanoparticles (AuNPs@GCE).

[0084] In a 10-fold PBS solution containing 1 μM ruthenium (II) tris(2,2'-bipyridyl) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, using the glassy carbon electrode modified with gold nanoparticles as the working electrode, platinum wire as the counter electrode, and silver / silver chloride as the reference electrode, electrochemiluminescence testing was carried out. Analysis was performed using the Xi'an Ruimai MPI-A type electrochemiluminescence analyzer. The high voltage of the photomultiplier tube was 500 V, the sampling frequency was 10 T / s, and the amplification factor was 3. The electrochemical method was cyclic voltammetry, the scanning range was -0.5 - 1.3 V, the scanning speed was 0.1 V / s, and the sensitivity was 1×10 -3 A / V. As Figure 8 shown, both electrodes had electrochemiluminescence peaks at around 0.9 V and 1.2 V. Among them, the electrochemiluminescence signal of the glassy carbon electrode modified with gold nanoparticles was weaker than that of the bare glassy carbon electrode.

[0085] In a 10-fold PBS solution containing 100 μM ruthenium (II) tris(2,2'-bipyridyl) chloride and 0.1 M tripropylamine, using AuNPs@GCE as the working electrode, platinum wire as the counter electrode, and silver / silver chloride as the reference electrode, electrochemiluminescence testing was carried out. Analysis was performed using the Xi'an Ruimai MPI-A type electrochemiluminescence analyzer. The high voltage of the photomultiplier tube was 300 V, the sampling frequency was 10 T / s, and the amplification factor was 3. The electrochemical method was cyclic voltammetry, the scanning range was -0.5 - 1.3 V, the scanning speed was 0.1 V / s, and the sensitivity was 1×10 -3 A / V, and scanned 20 cycles. This cyclic voltammetry test included "irreversible oxidation + reduction". As Figure 9 , as the gold nanoparticles were irreversibly oxidized to Au 2 O 3 and left the electrode surface at a voltage > 1.1 V, the electrode gradually restored the properties of the bare glassy carbon electrode, and the electrochemiluminescence signal increased with the number of cycles.

[0086] In a 10-fold PBS solution containing 100 μM ruthenium (II) tris(2,2'-bipyridyl) chloride and 0.1 M tripropylamine, electrochemical luminescence (ECL) measurements were performed with an AuNPs@GCE as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride electrode as the reference electrode. An MPI-A type ECL analyzer from Xi'an Ruimai was used for the analysis. The high voltage of the photomultiplier tube was 300 V, the sampling frequency was 10 T / s, and the amplification factor was 3. The electrochemical method was cyclic voltammetry with a scanning range of -0.5 to 0.9 V, a scanning rate of 0.1 V / s, and a sensitivity of 1×10 -3 A / V, and 10 cycles were scanned. This cyclic voltammetry test included "reversible oxidation + reduction". As Figure 10 , as the gold nanoparticles were reversibly oxidized to Au-O at voltages > 0.6 V and adsorbed on the electrode surface, and Au-O was reduced at voltages < 0 V, the ECL signal decreased slightly in the second cycle and then remained basically stable, indicating that the "reversible oxidation + reduction" strategy was effective, but it was still difficult to obtain a stable ECL signal by cyclic voltammetry.

[0087] In a 10-fold PBS solution containing 100 μM ruthenium (II) tris(2,2'-bipyridyl) chloride and 0.1 M tripropylamine, electrochemical luminescence (ECL) measurements were performed with an AuNPs@GCE as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride electrode as the reference electrode. An MPI-A type ECL analyzer from Xi'an Ruimai was used for the analysis. The high voltage of the photomultiplier tube was 300 V, the sampling frequency was 10 T / s, and the amplification factor was 3. The electrochemical method was cyclic voltammetry with a scanning range of 0.5 to 0.9 V, a scanning rate of 0.1 V / s, and a sensitivity of 1×10 -3 A / V, and 45 cycles were scanned. This cyclic voltammetry test included "irreversible oxidation + non-reduction". As Figure 11 , as the gold nanoparticles were reversibly oxidized to Au-O at voltages > 0.6 V and adsorbed on the electrode surface, the electrode surface was gradually blocked, and the electron transfer between the electrode and the ruthenium (II) tris(2,2'-bipyridyl) / tripropylamine in the solution became increasingly difficult, and the ECL signal decreased with the increase in the number of cycles.

[0088] The results showed that although the electrochemical strategy of "reversible oxidation + reduction" was helpful to some extent in inhibiting gold oxidation, the signal stability obtained by cyclic voltammetry was poor.

[0089] Take the prepared glassy carbon electrode modified with gold nanoparticles (AuNPs@GCE) to construct a sandwich-structured sensor on the electrode surface, where the antibody is labeled with a ruthenium terpyridine derivative through the EDC / NHS reaction. In a 10-fold PBS solution containing 0.1 M tripropylamine and 2% Twen20, using the sandwich-structured sensor as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride as the reference electrode for electrochemiluminescence testing. Analyze using the Xi'an Ruimai MPI-A type electrochemiluminescence analyzer, with a photomultiplier tube high voltage of 800 V, a sampling frequency of 10 T / s, and an amplification factor of 3. The electrochemical method is cyclic voltammetry, with a scanning range of -0.5 to 1.3 V, a scanning speed of 0.1 V / s, and a sensitivity of 1×10 -3 A / V. The electrochemiluminescence intensity-time image on the electrode surface is as Figure 12 , as the gold nanoparticles are irreversibly oxidized to Au 2 O 3 at a voltage greater than 1.1 V and leave the electrode surface, and the ruthenium terpyridine in the sandwich structure assembled on the gold nanoparticles also leaves the electrode surface, resulting in a significant decrease in the electrochemiluminescence signal.

[0090] Example 9

[0091] A pulsed voltage test method for inhibiting the oxidation of a gold electrode in electrochemical analysis, comprising the following steps:

[0092] a. Use metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm ɑ-Al 2 O 3 to polish the glassy carbon electrode in sequence, and then alternately ultrasonically clean it with ethanol and ultrapure water 3 times, 30 seconds each time. Insert the polished glassy carbon electrode into a 10-fold PBS buffer solution of 2 mM chloroauric acid, use the glassy carbon electrode as the working electrode, a silver / silver chloride electrode as the reference electrode, and a platinum wire as the counter electrode, and perform gold deposition on the surface of the glassy carbon electrode using a constant potential of -0.2 V. Control the deposition charge to be 1 mC to obtain a glassy carbon electrode modified with gold nanoparticles (AuNPs@GCE). In a 10-fold PBS solution containing 1 μM ruthenium terpyridine chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, use the glassy carbon electrode modified with gold nanoparticles as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride as the reference electrode for electrochemiluminescence testing.

[0093] b. Oxidation stage: As Figure 13 , apply a pulsed constant voltage of 0.01 s and 0.9 V, and an oxidation reaction occurs on the surface of the glassy carbon electrode modified with gold nanoparticles to generate an electrochemiluminescence signal;

[0094] c. Equilibrium stage: Apply a voltage of 0 V for 0.5 s to return the system to the equilibrium state;

[0095] d. Reduction stage: Apply a constant pulse voltage of 0.5 s and -0.5 V. A reduction reaction occurs on the surface of the glassy carbon electrode modified with gold nanoparticles, which is used to reduce the reversibly formed gold oxide during the oxidation stage;

[0096] e. Equilibrium stage: Apply a voltage of 0 V for 2 s to return the system to the equilibrium state;

[0097] f. Repeat steps b - e 15 times until sufficient data for analyzing the signal quality is obtained.

[0098] Take the average light intensity value of the last 3 ms of each oxidation pulse as the signal value of this cycle. As Figure 14 shown, this pulsed voltage method is used to test the gold electrode system with surface - immobilized ruthenium terpyridine and achieves good results (relative standard deviation RSD = 5.86%).

[0099] Example 10

[0100] A pulsed voltage test method for inhibiting the oxidation of a gold electrode in electrochemical analysis, comprising the following steps:

[0101] a. Polish the gold electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α - Al 2 O 3 and then ultrasonically clean it alternately with ethanol and ultrapure water 3 times, 30 seconds each time. In a 10 - fold PBS solution containing 1 μM ruthenium terpyridine chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, use the gold electrode as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode as the reference electrode to conduct electrochemiluminescence testing.

[0102] b. Oxidation stage: Apply a constant pulse voltage of 1 s and 1.1 V. An oxidation reaction occurs on the surface of the gold electrode, which is used to generate an electrochemiluminescence signal;

[0103] c. Equilibrium stage: Apply a voltage of 0 V for 2 s to return the system to the equilibrium state;

[0104] d. Reduction stage: Apply a constant pulse voltage of 0.3 s and 0.5 V. A reduction reaction occurs on the surface of the gold electrode, which is used to reduce the reversibly formed gold oxide during the oxidation stage;

[0105] e. Equilibrium stage: Apply a voltage of 0 V for 0.5 s to return the system to the equilibrium state;

[0106] f. Repeat steps b - e 20 times until sufficient data for analyzing the signal quality is obtained.

[0107] Example 11

[0108] A pulsed voltage test method for inhibiting the oxidation of a gold electrode in electrochemical analysis, comprising the following steps:

[0109] a. Polish the gold electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al 2 O 3 and then ultrasonically clean it alternately with ethanol and ultrapure water 3 times, 30 seconds each time. In a 10-fold PBS solution containing 1 μM ruthenium(III) tris(bipyridine) chloride and 0.1 M tripropylamine, with a pH of 7.2 - 7.4, use the gold electrode as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride as the reference electrode to conduct electrochemiluminescence testing.

[0110] b. Oxidation stage: Apply a pulsed constant voltage of 1.0 V for 0.5 s, and an oxidation reaction occurs on the surface of the gold electrode to generate an electrochemiluminescence signal;

[0111] c. Equilibrium stage: Apply a voltage of 0 V for 1 s to return the system to the equilibrium state;

[0112] d. Reduction stage: Apply a pulsed constant voltage of 0.2 V for 1 s, and a reduction reaction occurs on the surface of the gold electrode to reduce the reversibly formed gold oxide during the oxidation stage;

[0113] e. Equilibrium stage: Apply a voltage of 0 V for 0.5 s to return the system to the equilibrium state;

[0114] f. Repeat steps b - e 8 times until sufficient data for analyzing the signal quality is obtained.

[0115] Example 12

[0116] A pulsed voltage test method for inhibiting the oxidation of a gold electrode in electrochemical analysis, comprising the following steps:

[0117] a. Polish the gold electrode successively with metallographic sandpaper, 1 μm, 0.3 μm, and 0.05 μm α-Al 2 O 3 and then ultrasonically clean it alternately with ethanol and ultrapure water 3 times, 30 seconds each time. In a 10-fold PBS solution containing 100 mM potassium ferricyanide / potassium ferrocyanide, with a pH of 7.2 - 7.4, use the gold electrode as the working electrode, a platinum wire as the counter electrode, and a silver / silver chloride as the reference electrode to conduct electrochemical testing.

[0118] b. Oxidation stage: Apply a pulsed constant voltage of 0.6 V for 0.5 s, and an oxidation reaction occurs on the surface of the gold electrode to generate an oxidation current signal;

[0119] c. Equilibrium stage: Apply a voltage of 0 V for 1 s to return the system to the equilibrium state;

[0120] d. Reduction stage: Apply a constant voltage pulse of 0.2 V for 1 s. A reduction reaction occurs on the surface of the gold electrode, which is used to reduce the reversibly formed gold oxide during the oxidation stage;

[0121] e. Equilibrium stage: Apply a voltage of 0 V for 0.5 s to bring the system back to the equilibrium state;

[0122] f. Repeat steps b - e 10 times until sufficient data for analyzing the signal quality is obtained.

Claims

1. A pulsed voltage test method for suppressing the oxidation of a gold electrode in electrochemical analysis, characterized in that, it comprises the following steps: Step 1, put a gold electrode or a glassy carbon electrode modified with gold nanoparticles, a counter electrode, and a reference electrode into the solution to be analyzed, and conduct electrochemical testing; Step 2, apply a pulsed constant voltage of 0.01 - 1 s and 0.6 - 1.1 V, and an oxidation reaction occurs on the surface of the gold electrode or the glassy carbon electrode modified with gold nanoparticles to generate an electrochemical signal; Step 3, apply a pulsed constant voltage of 0.3 - 1 s and -0.5 - 0.5 V, and a reduction reaction occurs on the surface of the gold electrode or the glassy carbon electrode modified with gold nanoparticles to reduce the reversibly generated gold oxide in the oxidation stage; Step 4, repeat Step 2 - Step 3 more than 5 times to obtain sufficient data for analyzing the signal quality, avoid the oxidation influence of the gold electrode or the glassy carbon electrode modified with gold nanoparticles, and obtain an electrochemical or optical signal with a quality higher than that of the non-pulsed method; the solution to be analyzed is a 10-fold phosphate buffer solution of ruthenium(III) chloride tris(bipyridine) and tripropylamine; after Step 2, apply a voltage of 0 V for 0.5 - 2 s to return the system to the equilibrium state; after Step 3, apply a voltage of 0 V for 0.5 - 2 s to return the system to the equilibrium state.

2. The pulsed voltage test method for suppressing the oxidation of a gold electrode in electrochemical analysis according to claim 1, characterized in that: the counter electrode is a platinum wire.

3. The pulsed voltage test method for suppressing the oxidation of a gold electrode in electrochemical analysis according to claim 1, characterized in that: the reference electrode is a silver chloride electrode or a saturated calomel electrode.

4. The pulsed voltage test method for suppressing the oxidation of a gold electrode in electrochemical analysis according to claim 1, characterized in that: the pH of the 10-fold phosphate buffer solution is 7.2 - 7.4.

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