A method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence

Abstract

The present invention discloses a method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence, which belongs to the field of analytical chemistry. The method comprises: S1: fixing a microsphere with a luminescent molecule modified on its surface to an electrode surface as a working electrode; S2: dissolving the tertiary amine molecule to be measured in an electrolyte solution to obtain a test solution; S3: immersing the working electrode, reference electrode, and counter electrode in the test solution, and applying a potential at the same time, causing the luminescent molecules on the surface of the microspheres to emit light through electrochemical and chemical reactions, and then stopping the application of the potential, and recording the luminescent signal during this process; the time required for the light intensity to decay to the background signal value is the lifetime of the cation radical corresponding to the tertiary amine molecule to be measured. The method of the present invention is novel, simple in apparatus, and short in measurement time, and is of great significance for studying the mechanism of electrochemiluminescence, developing new and efficient co-reactants, and improving the performance of electrochemiluminescence detection.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence. Background Art

[0002] Electrochemiluminescence is a dark field light radiation caused by the reaction on the electrode surface. A certain electrochemical signal (voltage or current) is applied to the system containing the luminescent molecule and the co-reactant through the electrode. The intermediates produced by the reaction of the luminescent molecule and the co-reactant on the electrode surface generate excited luminescent molecules through further chemical reactions. The excited luminescent molecules transition back to the ground state and generate light radiation. Therefore, electrochemiluminescence has the advantages of no light excitation, low background, and high sensitivity. Tertiary amine molecules are one of the most common co-reactants. Tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)3 2+ The system with ) and tri-n-propylamine (TPrA) as the luminescent molecule and co-reactant is the most widely used electrochemiluminescence system, and the electrochemiluminescence immunoassay based on this system has achieved high-sensitivity detection of more than one hundred disease marker target molecules in clinical practice.

[0003] The principle of the electrochemiluminescence immunoassay is as follows: the antigen and antibody form a sandwich structure through immune reaction, and one of the antibodies is labeled with Ru(bpy)3 2+ ; The immune sandwich structure is separated to the electrode surface by magnetic microspheres with a diameter of 2.8 μm, and then TPrA is added. When a suitable voltage is applied, the luminescent probe can generate a luminescent signal. 2+ It is fixed on the surface of magnetic microspheres, so only TPrA undergoes electrochemical reaction on the electrode surface, and the generated tri-n-propylamine cation radical (TPrA +· ) diffuses to the surface of magnetic microspheres and Ru(bpy)3 2+ The reaction generates an excited state and produces a light signal. Therefore, in electrochemiluminescence immunoassay, TPrA +· The life of the TprA has a huge impact on the performance of the detection. A longer life can make the TprA +· Diffusion over longer distances, with more Ru(bpy)3 2+ The reaction produces a stronger light signal, thereby improving the sensitivity of the detection.

[0004] However, the measurement of the lifetime of amine cation radicals is very difficult because the lifetime of the radicals is very short (in the order of microseconds) and the reaction of cation radicals generated by the oxidation of tertiary amine molecules is very complex. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence. This method, with its simple measurement apparatus and short measurement time, is of great significance for studying the mechanism of electrochemiluminescence, developing new and efficient co-reactants, and improving electrochemiluminescence detection performance.

[0006] The specific technical solutions adopted in the present invention are as follows:

[0007] The present invention provides a method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence, which is as follows:

[0008] S1: Fix the microspheres modified with luminescent molecules on the electrode surface as the working electrode;

[0009] S2: dissolving the tertiary amine molecule to be tested in an electrolyte solution to obtain a test solution;

[0010] S3: Immerse the working electrode, reference electrode and counter electrode in the solution to be tested, and apply potential at the same time to make the luminescent molecules on the surface of the microspheres emit light through electrochemical reactions and chemical reactions. Then stop applying the potential and record the luminescent signal during this process; the time required for the light intensity to decay to the background signal value is the lifetime of the cationic free radical corresponding to the tertiary amine molecule to be tested.

[0011] Preferably, the luminescent molecule is one of tris(2,2'-bipyridine)ruthenium(II) and its derivatives.

[0012] Preferably, the microspheres are polystyrene microspheres, silica microspheres or magnetic microspheres, with a diameter of 1-10 μm.

[0013] Preferably, the luminescent molecule is modified onto the surface of the microspheres by an amidation reaction, and the specific process is as follows:

[0014] The surface-aminated microspheres react with tris(2,2'-bipyridine)ruthenium(II) modified with carboxyl functional groups or the surface-carboxylated microspheres react with tris(2,2'-bipyridine)ruthenium(II) modified with amino functional groups, and the carboxyl groups are activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0015] Preferably, in step S1, the microspheres with luminescent molecules modified on the surface are fixed to the surface of the electrode by drop coating; the electrode is a gold electrode, a platinum electrode, a glassy carbon electrode or an indium tin oxide electrode.

[0016] Preferably, the electrolyte solution is a 0.2 M phosphate buffer solution with a pH of 7.4; and the concentration of the tertiary amine molecules in the test solution is 100 mM.

[0017] Preferably, in step S3, a potential of 1.0 V to 1.5 V is applied by chronoamperometry; the reference electrode and the counter electrode are a silver / silver chloride electrode and a platinum electrode, respectively.

[0018] Preferably, the luminescence signal is recorded by a photon counter or a photomultiplier tube, and the photon counter or the photomultiplier tube realizes synchronous measurement with the electrical signal through a TTL external trigger signal, and the time interval for measuring the luminescence signal is 50 μs.

[0019] Ru(bpy)3 2+ When fixed on the surface of microspheres, the reaction mechanism of electrochemiluminescence is as follows:

[0020] R→R +· +e - (1)

[0021] R +· →R · +H + (2)

[0022]

[0023]

[0024]

[0025] R, R +· 、R · and P are the reaction byproducts of tertiary amine, tertiary amine cation radical, tertiary amine radical and tertiary amine radical respectively. R is oxidized to R on the electrode surface. +· (Equation 1), R +· Deprotonation generates R · (Equation 2). Then, R · Ru(bpy)3 2+ Reduction to Ru(bpy)3 + (Equation 3), R +· Ru(bpy)3 + Oxidation to excited state Ru(bpy)3 2+* (Equation 4). Finally, Ru(bpy)3 2+* It transitions back to the ground state and radiates photons (Equation 5). When a potential is applied to the system, excited state luminescent molecules are generated and photons are radiated according to Equations 1-5. When the potential is stopped, the electrochemical reaction on the electrode surface terminates, that is, Equation 1 terminates, and excited state luminescent molecules are generated and photons are radiated according to Equations 2-5. 2+* The lifetime is several hundred nanoseconds, much lower than R +· The lifetime of the light emitting diode is (in microseconds), so the duration of the light emitting diode after the potential is stopped is R +·The duration of its existence, that is, its lifespan.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The method provided by the present invention is rapid and convenient; 2. The instrumentation required for the method is simple and inexpensive; 3. The method is a novel method for measuring the lifetime of tertiary amine cation radicals; 4. The method is of great significance for studying the mechanism of electrochemiluminescence, developing new and efficient co-reactants, and improving the performance of electrochemiluminescence detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Modification of aminopolystyrene microspheres with Ru(bpy)3 2+ Schematic diagram of;

[0029] Figure 2 Schematic diagram of the device for synchronous measurement of electrochemical signals and optical signals;

[0030] Figure 3 is a curve showing the change of electrochemiluminescence signal over time in Example 1;

[0031] Figure 4 is a curve showing the change of electrochemiluminescence signal over time in Example 2;

[0032] Figure 5 This is a curve showing the electrochemiluminescence signal changing with time in Example 3. DETAILED DESCRIPTION

[0033] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention may be combined accordingly, provided that there is no conflict between them.

[0034] The present invention provides a method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence, which is specifically as follows:

[0035] (1) The luminescent molecules are modified onto the surface of the microspheres, and the microspheres are fixed to the surface of the electrode, which serves as the working electrode.

[0036] In practical applications, the luminescent molecule can be tris(2,2'-bipyridine)ruthenium(II) and one of its derivatives; the microspheres have a diameter of 1-10 μm and are polystyrene, silica, or magnetic. The luminescent molecule is modified onto the microsphere surface through an amidation reaction, whereby the surface-amino-modified microspheres react with tris(2,2'-bipyridine)ruthenium(II) modified with a carboxyl functional group, or the surface-carboxylated microspheres react with tris(2,2'-bipyridine)ruthenium(II) modified with an amino functional group, and the carboxyl groups are activated using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

[0037] In practical applications, the microspheres modified with luminescent molecules are fixed to the surface of an electrode by drop coating. The electrode can be a gold electrode, a platinum electrode, a glassy carbon electrode or an indium tin oxide electrode.

[0038] (2) The tertiary amine molecule to be tested is dissolved in an electrolyte solution, and the tertiary amine molecule is a co-reactant of electrochemiluminescence.

[0039] In practical applications, the electrolyte solution is preferably a 0.2 M phosphate buffer solution with a pH of 7.4; and the concentration of the tertiary amine molecules in the test solution is 100 mM.

[0040] (3) The working electrode, reference electrode, and counter electrode are immersed in the electrolyte solution, and a potential is applied to the electrode system (composed of the working electrode, reference electrode, and counter electrode) to make the luminescent molecules on the surface of the microspheres emit light through electrochemical and chemical reactions.

[0041] In practical applications, the potential is applied by chronoamperometry, and the applied potential range is 1.0V-1.5V depending on the electrode used; the reference electrode and the counter electrode are silver / silver chloride (saturated KCl) electrode and platinum electrode, respectively.

[0042] (4) Stop applying the potential; and simultaneously, record the luminescence signal by a photon counter or a photomultiplier tube during the entire process of applying the potential and after stopping the application of the potential.

[0043] In practical applications, the photon counter or photomultiplier tube realizes synchronous measurement with the electrical signal through a TTL external trigger signal, and the time interval for measuring the luminescence signal is 50 μs.

[0044] (5) The time required for the light intensity to decay to the background signal value is the lifetime of the cationic radical corresponding to the tertiary amine molecule to be measured.

[0045] The method of the present invention will be specifically described and its effects will be verified by way of examples below.

[0046] Example 1

[0047] like Figure 1As shown, amino polystyrene microspheres (PSB) with a diameter of 3.8 μm and carboxylated Ru(bpy)3 2+ Mixed, surface modified Ru(bpy)3 was obtained by amidation reaction 2+ A solution containing Ru-PSBs was added dropwise to the surface of an indium tin oxide (ITO) electrode. After the solvent evaporated, the Ru-PSBs were adsorbed and fixed to the electrode surface. A three-electrode system was used, with an ITO electrode, a platinum wire, and a silver / silver chloride (saturated with KCl) electrode serving as the working, counter, and reference electrodes. Chronoamperometry was used to apply a voltage of +1.5 V. The electrolyte solution was a 0.2 M solution of PB containing 100 mM tri-n-propylamine.

[0048] like Figure 2 As shown, the working electrode is placed horizontally, and a square polymer film with a circular hole in the middle is pasted on the surface of the working electrode as an electrolytic cell. The electrolyte is dripped into the circular hole of the polymer, and the reference electrode and platinum wire are inserted into the electrolyte. The potential is applied through the electrochemical workstation, and the light signal is recorded with a photon counting detector (located above the working electrode) and a photon counter, and the electrochemical and optical measurements are synchronized by TTL signals. After the potential is stopped, the time required for the light intensity to decay to the background is the lifetime of the tertiary amine cation radical. Figure 3 As shown, the lifetime of the tri-n-propylamine cation radical can be obtained from the curve of the change of luminescence intensity over time to be 241 μs, which is consistent with the lifetime of the tri-n-propylamine cation radical reported in the literature (J.Am.Chem.Soc.2002,124,14478-14485), proving the reliability and accuracy of the present invention.

[0049] Example 2

[0050] Amino polystyrene microspheres (PSB) with a diameter of 3.8 μm and carboxylated Ru(bpy)3 2+ Mixed, surface modified Ru(bpy)3 was obtained by amidation reaction 2+ A solution containing Ru-PSBs was added dropwise to the surface of a glassy carbon electrode. After the solvent evaporated, the Ru-PSBs were adsorbed and fixed to the electrode surface. A three-electrode system was used, with a glassy carbon electrode, platinum wire, and a silver / silver chloride (saturated with KCl) electrode serving as the working, counter, and reference electrodes. A voltage of +1.2 V was applied using chronoamperometry. The electrolyte solution was a 0.2 M solution of PB containing 100 mM 2-(dibutylamino)ethanol.

[0051] like Figure 2As shown, the working electrode is placed horizontally, and a square polymer film with a circular hole in the middle is pasted on the surface of the working electrode to serve as an electrolytic cell. The electrolyte is dripped into the circular hole of the polymer, and the reference electrode and platinum wire are inserted into the electrolyte. The potential is applied through the electrochemical workstation, and the light signal is recorded with a photon counting detector and a photon counter, and the electrochemical and optical measurements are synchronized by TTL signals. After the potential is stopped, the time required for the light intensity to decay to the background is the lifetime of the tertiary amine cation radical. Figure 4 As shown, the lifetime of the 2-(dibutylamino)ethanol cation radical can be obtained as 134 μs through the curve of the change of luminescence intensity over time.

[0052] Example 3

[0053] Silica microspheres with a diameter of 2.8 μm and Ru(bpy)3 2+ Mixed, silica microspheres with negative charge on the surface, Ru(bpy)3 2+ The Ru(bpy)3 2+ Modified silica microspheres were added dropwise to the surface of a glassy carbon electrode. After the solvent evaporated, the microspheres were adsorbed and fixed to the electrode surface. A three-electrode system was used, with a glassy carbon electrode, platinum wire, and a silver / silver chloride (saturated with KCl) electrode serving as the working, counter, and reference electrodes. Chronoamperometry was used to apply a voltage of +1.2 V. The electrolyte solution was a 0.2 M solution of PB containing 100 mM N-butyldiethanolamine.

[0054] like Figure 2 As shown, the working electrode is placed horizontally, and a square polymer film with a circular hole in the middle is pasted on the surface of the working electrode to serve as an electrolytic cell. The electrolyte is dripped into the circular hole of the polymer, and the reference electrode and platinum wire are inserted into the electrolyte. The potential is applied through the electrochemical workstation, and the light signal is recorded with a photon counting detector and a photon counter, and the electrochemical and optical measurements are synchronized by TTL signals. After the potential is stopped, the time required for the light intensity to decay to the background is the lifetime of the tertiary amine cation radical. Figure 5 As shown, the lifetime of the 2-(dibutylamino)ethanol cation radical is 100 μs according to the curve of the change of luminescence intensity over time.

[0055] The method of the present invention is novel, the device is simple, and the measurement time is short, which is of great significance for studying the electrochemiluminescence mechanism, developing new and efficient co-reactants, and improving the electrochemiluminescence detection performance.

[0056] The embodiment described above is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. A method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence, characterized in that: The details are as follows: S1: Fix the microspheres modified with luminescent molecules on the electrode surface as the working electrode; S2: dissolving the tertiary amine molecule to be tested in an electrolyte solution to obtain a test solution; S3: Immersing the working electrode, reference electrode, and counter electrode in the solution to be tested, applying a potential simultaneously, causing the luminescent molecules on the surface of the microspheres to emit light through electrochemical and chemical reactions, then stopping the application of the potential, and recording the luminescent signal during this process; the time required for the light intensity to decay to the background signal value is the lifetime of the cation radical corresponding to the tertiary amine molecule to be tested; The luminescent molecule is one of tris(2,2'-bipyridine)ruthenium(II) and its derivatives; The microspheres are polystyrene microspheres, silica microspheres or magnetic microspheres, and have a diameter of 1-10 μm.

2. The method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence according to claim 1, characterized in that: The luminescent molecules are modified onto the surface of the microspheres through an amidation reaction, and the specific process is as follows: The surface-aminated microspheres react with tris(2,2'-bipyridine)ruthenium(II) modified with carboxyl functional groups or the surface-carboxylated microspheres react with tris(2,2'-bipyridine)ruthenium(II) modified with amino functional groups, and the carboxyl groups are activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

3. The method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence according to claim 1, characterized in that: In the step S1, the microspheres with luminescent molecules modified on the surface are fixed to the surface of the electrode by drop coating; the electrode is a gold electrode, a platinum electrode, a glassy carbon electrode or an indium tin oxide electrode.

4. The method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence according to claim 1, characterized in that: The electrolyte solution is a 0.2 M phosphate buffer solution with a pH of 7.4; the concentration of the tertiary amine molecules in the test solution is 100 mM.

5. The method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence according to claim 1, characterized in that: In step S3, a potential of 1.0 V to 1.5 V is applied by chronoamperometry; the reference electrode and the counter electrode are a silver / silver chloride electrode and a platinum electrode, respectively.

6. The method for measuring the lifetime of tertiary amine cation radicals based on time-resolved electrochemiluminescence according to claim 1, characterized in that: The luminescence signal is recorded by a photon counter or a photomultiplier tube, and the photon counter or the photomultiplier tube realizes synchronous measurement with the electrical signal through a TTL external trigger signal. The time interval of the luminescence signal measurement is 50 μs.

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