CN108872209A - Alkaline phosphatase assay method based on nanogold cluster electrogenerated chemiluminescence probe - Google Patents

Abstract

The invention discloses an alkaline phosphatase assay method based on a nano-gold cluster electrochemiluminescence probe. The alkaline phosphatase determination method of the nano-gold cluster electrochemiluminescence probe is to prepare a high-quantum-yield nano-gold cluster electrochemiluminescence probe using a reduction method, and use manganese dioxide nanomaterials as the electrochemiluminescence quenching method. The reagent uses alkaline phosphatase to selectively hydrolyze 2-phosphate-L-ascorbic acid trisodium salt to produce redox reaction of ascorbic acid and manganese dioxide to restore the electrochemiluminescence signal, and realize the determination of alkaline phosphatase content. In the concentration range of 1.0×10 ^‑4 ~2.0×10 ² U/L, the logarithm value of alkaline phosphatase concentration has a linear relationship with the change value of relative electrochemiluminescence intensity, and the detection limit is 1.5×10 ^‑5 U/L . The method has the advantages of simple operation, low cost, high sensitivity, good reproducibility, etc., can be used for the determination of alkaline phosphatase in actual samples, and has good clinical application prospects.

Description

Alkaline Phosphatase Determination Method Based on Nano-gold Cluster Electrochemiluminescence Probe

technical field

The invention relates to an alkaline phosphatase assay method of a nano-gold cluster electrochemiluminescent probe, which belongs to the field of analytical chemistry and nanotechnology.

Background technique

Alkaline phosphatase (Alkaline phosphates) is a plasma membrane-bound glycoprotein, which can catalyze the hydrolysis of molecules such as nucleic acids, proteins, and phospholipids to remove the phosphate groups in them, and play a role in biological processes such as gene transfer, cell metabolism, and nervous system. plays a key role. Alkaline phosphatase is widely distributed in nature (except some higher plants), and widely exists in various organs of the human body, especially in the liver and bones, and its content is relatively high, while the content in the placenta and kidney is relatively small . Studies have found that the content of alkaline phosphatase is closely related to the occurrence and development of various diseases. For example, in the serum of patients with osteomalacia, liver dysfunction, and obstructive jaundice, alkaline phosphatase is highly expressed. Therefore, in clinical testing, alkaline phosphatase is used as a biomarker for many diseases. As a key biomarker for the diagnosis of many diseases, alkaline phosphatase is one of the most commonly measured enzymes in clinical practice. At present, the methods used to detect alkaline phosphatase, such as colorimetry, fluorescence analysis, chemiluminescence, surface-enhanced Raman spectroscopy, and electrochemical methods, have low sensitivity and accuracy, cumbersome operation, and high cost. High, long detection time and other shortcomings, which greatly limit its application in clinical diagnosis. Therefore, it is of great practical significance to establish a simple, rapid and highly sensitive new method for the detection of alkaline phosphatase content.

Electrochemiluminescence (ECL) bioanalysis is a new analysis method developed in recent years, and it is the latest product of the combination of chemiluminescence, electrochemistry, bioanalysis, microelectronics technology and sensing technology. The analysis method has many excellent performance characteristics such as simple operation, rapidity, good selectivity, wide linear range, and easy control, and ECL technology does not require excitation light source and low background signal, which can not only effectively avoid photobleaching and Background interference problem, and more sensitive than conventional fluorescence analysis, has been widely used in the determination of clinical diagnosis, medicine, immunity, food quality and other substances. In recent years, the electrochemiluminescence analysis method based on quantum dots or nanoclusters has been used in biology, medicine, and the environment have been widely used. As a new type of nanoluminescent body, gold nanoclusters have the characteristics of non-toxicity, good water solubility, large specific surface area, mild preparation conditions, easy surface modification, and special photoelectric properties. They have been widely used in biomedicine, clinical analysis, sensing detection and catalysis. Widely used in other fields. Our research group has successfully realized the preparation of electrochemiluminescence probes of nano-gold clusters with high quantum yield through nanoscale regulation, and established a series of new high-efficiency electrochemiluminescence life analysis platforms on this basis.

The present invention uses nano-gold clusters as electrochemiluminescent probes, manganese dioxide nanosheets as quenching agents, and based on alkaline phosphatase to selectively hydrolyze ascorbic acid and manganese dioxide produced by 2-phosphate-L-ascorbic acid trisodium salt. The electrochemiluminescence signal was restored by the oxidation-reduction reaction, and a new method for the determination of alkaline phosphatase with high sensitivity, high selectivity and rapidity was established.

Contents of the invention

The purpose of the present invention is to provide an alkaline phosphatase assay method using nano-gold clusters as electrochemiluminescent probes.

In order to achieve the above object, the present invention adopts the following technical scheme: a method for measuring alkaline phosphatase based on nano-gold cluster electrochemiluminescence probe, which is characterized in that: first, nano-gold clusters are modified on the electrode surface, and high Quantum yield nano-gold cluster electrochemiluminescence probe, and further modify manganese dioxide nanomaterials on the electrode surface as an electrochemiluminescence quencher; add alkaline phosphatase to 2-phospho-L-ascorbic acid tris In the sodium salt solution, alkaline phosphatase selectively hydrolyzes 2-phosphate-L-ascorbic acid trisodium salt to produce ascorbic acid, and then immerses the modified electrode in the above reaction solution, and the generated ascorbic acid undergoes a redox reaction with manganese dioxide; the modified electrode is collected The electrochemiluminescent signal, according to the change of the electrochemiluminescent signal, realizes the determination of alkaline phosphatase.

The reduction method to prepare high quantum yield nano-gold cluster electrochemiluminescent probes is prepared by the following electrochemical reduction method or chemical reduction method:

(1) Electrochemical reduction method: a three-electrode system was adopted, with a nano-gold cluster modified glassy carbon electrode as the working electrode, a platinum wire electrode as the counter electrode, and Ag/AgCl as the reference electrode, and the above electrodes were inserted into 0.1 mol/L, pH 7.4 In the phosphate buffer solution, apply a negative potential voltage in the range of -1.4 V to -2 V, and perform a constant potential reduction treatment for 5 min to 1 h to obtain an electrochemiluminescence nano-gold cluster probe;

(2) Chemical reduction method: soak the nano-gold cluster modified glassy carbon electrode in 0.1-1 mol/L sodium borohydride solution for 5-30 min to react to obtain the nano-gold cluster probe-modified electrode prepared by the chemical reduction method.

The nano-gold clusters are functionally modified nano-gold clusters, using N-acetylated-L-cysteine-nano-gold clusters or bovine serum albumin-nano-gold clusters.

The method for modifying manganese dioxide nanomaterials on the electrode surface is an electrochemical deposition method: a three-electrode system is used, and the electrode modified by the reduction method to treat the nano-gold cluster is used as the working electrode, the platinum wire electrode is used as the counter electrode, and Ag/AgCl is used as the reference Electrode, using It method, in 1:1 V/V KMnO ₄ -H ₂ SO ₄ solution, electrochemically deposit manganese dioxide, the potential is -0.1 V~-0.5V, the deposition time is 100 s~600 s, Clean and blow dry with nitrogen.

The method for collecting the electrochemiluminescent signal of the modified electrode is as follows: a three-electrode system is used for testing, the modified electrode is used as the working electrode, the platinum wire electrode is used as the counter electrode, Ag/AgCl is used as the reference electrode, and the buffer solution is a phosphate buffer solution Or Tris-HCl buffer solution, the electrolyte used is KCl or KNO ₃ ; the above electrode is inserted into the buffer solution containing persulfate ion co-reactant, and the electrochemiluminescence test is carried out by step pulse method, the initial potential is 0 V, and the pulse The time is 10 s, the termination potential is -2 V, the pulse time is 1 s, and the high voltage of the photomultiplier tube is set to 600-800 V to detect the electrochemiluminescence radiation signal.

The pH of the oxidation-reduction reaction between the produced ascorbic acid and manganese dioxide is 8.0, and the reaction time is 4 minutes.

The pH value of the phosphate buffer solution or Tris-HCl buffer solution for collecting the electrochemiluminescent signal of the modified electrode is 7.4, and the concentration of persulfate ion is 0.1 mol/L.

The change value of electrochemiluminescence intensity and the log value of ascorbic acid concentration showed a good linear relationship in the range of 1.0×10 ^-4 ~2.0×10 ² U/L, and the detection limit was 1.5×10 ^-5 U/L.

Specifically, in order to achieve the above purpose, the present invention adopts the following specific technical solutions: modify nano-gold clusters on glassy carbon electrodes, prepare nano-gold clusters electrochemiluminescent probes with high quantum yields by reduction method, and then Electrodeposition of manganese dioxide nanomaterials; alkaline phosphatase is added to 2-phosphate-L-ascorbic acid trisodium salt solution, alkaline phosphatase selectively hydrolyzes 2-phosphate-L-ascorbic acid trisodium salt to produce ascorbic acid, and the two The manganese oxide/nano-gold cluster modified electrode was immersed in the above reaction solution; the electrochemiluminescence signal of the modified electrode was collected, and the electrochemiluminescence signal was recovered by redox reaction between ascorbic acid produced in the reaction solution and manganese dioxide on the surface of the electrode. A change in the electrochemiluminescent signal enables the determination of alkaline phosphatase.

The above nano-gold cluster probes were prepared by the following methods: (1) Electrochemical reduction method: a three-electrode system was used for reduction, with nano-gold cluster modified glassy carbon electrode as the working electrode, platinum wire electrode as the counter electrode, and Ag/AgCl as the reference electrode. Compared with the electrode, insert the above electrode into the buffer solution, apply different negative potential voltages, and perform constant potential reduction treatment to obtain electrochemiluminescence nano-gold cluster probes; (2) chemical reduction method: soak the nano-gold cluster modified glassy carbon electrode in The sodium borohydride solution of a certain concentration is reacted for a certain period of time to obtain a nano-gold cluster probe-modified electrode prepared by a chemical reduction method.

The nano-gold cluster materials are functionally modified nano-gold clusters, such as: N-acetylated-L-cysteine-nano-gold clusters, bovine serum albumin-nano-gold clusters, and the like.

The method for electrodepositing manganese dioxide nanomaterials on the electrode surface is as follows: using a three-electrode system, using a reduction method to treat the nano-gold cluster modified electrode as the working electrode, the platinum wire electrode as the counter electrode, and Ag/AgCl as the reference electrode, Using IT method, in KMnO ₄ -H ₂ SO ₄ (1:1 V/V) solution, the manganese dioxide nanomaterial modified electrode was prepared by constant potential deposition.

The electrochemiluminescent signal is collected by the following method: a three-electrode system is used for testing, a modified glassy carbon electrode is used as a working electrode, a platinum wire electrode is used as a counter electrode, and Ag/AgCl is used as a reference electrode. in the buffered solution of the reagents. The step pulse method was adopted, and the high voltage of the photomultiplier tube was set at 600 V to 800 V to detect the electrochemiluminescent signal generated on the surface of the working electrode.

A method for measuring ascorbic acid based on nano-gold clusters as electrochemiluminescent probes of the present invention comprises the following steps: ₁ ) polishing and polishing the glassy carbon electrode with _Al2O3 powder in sequence until it reaches a smooth mirror surface, and then sequentially Put in HNO ₃ solution, absolute ethanol, and deionized water for ultrasonic cleaning, and blow dry with N ₂ ; 2) Add nano-gold cluster probe solution on the surface of the treated glassy carbon electrode, and dry at room temperature to obtain nano-gold clusters Probe-modified glassy carbon electrodes, prepared electrochemiluminescent probes with high quantum yield nano-gold clusters by reduction method, and further prepared manganese dioxide nanomaterials/nano-gold cluster modified electrodes; 3) Take different concentrations of alkaline phosphoric acid The enzyme solution is added to the 2-phospho-L-ascorbic acid trisodium salt solution, mixed evenly, and then the manganese dioxide/nano gold cluster probe modified glassy carbon electrode is put into the above solution for soaking. After taking it out, rinse it with double distilled water, dry it with N ₂ and use it for later use; 4) Use a three-electrode system for testing, use the manganese dioxide/nano-gold cluster probe-modified glassy carbon electrode as the working electrode after the above reaction, and the platinum wire electrode as For the counter electrode, Ag/AgCl is the reference electrode. Insert the above electrode into the buffer solution containing the co-reactant to detect its electrochemiluminescence signal value; adopt the step pulse method, and set the high voltage of the photomultiplier tube to 600 V ~ 800 V , to detect the electrochemiluminescence signal generated on the surface of the working electrode; 4) plotting the change value of the electrochemiluminescence signal against the logarithm value of the concentration of alkaline phosphatase to obtain a standard curve, when the concentration of alkaline phosphatase is 1.0×10 ^-4 The logarithmic value of ascorbic acid concentration in the range of ~2.0×10 ² U/L has a good linear relationship with the value of electrochemiluminescence intensity, and the detection limit is 1.5×10 ^-5 U/L.

The above co-reactant is persulfate ion, the concentration range is 0.01-1 mol/L, preferably 0.1 mol/L.

The buffer solution is a phosphate buffer solution or a Tris-HCl buffer solution, and the electrolyte added in the buffer solution is KCl or KNO ₃ at a concentration of 0.01-1 mol/L.

The concrete technical scheme that the present invention adopts is as follows:

(1) Preparation of N-acetyl-L-cysteine-nano-gold clusters

The synthesis steps of N-acetyl-L-cysteine-nano-gold clusters are as follows: add sodium hydroxide with a concentration of 0.1-0.8 mol/L and chloroauric acid solution with a concentration of 0.01-0.1 g/L to a concentration of 0.02- 0.18 mol/L N-acetyl-L-cysteine solution, mix well and place in a constant temperature water bath at 20-70 ℃ for constant temperature reaction for 0-3.5 hours. After the reaction is completed, dialysis and purification treatment is carried out to obtain an aqueous solution of N-acetyl-L-cysteine-nano-gold clusters, and after freeze-drying, N-acetyl-L-cysteine-nano-gold clusters material powder can be obtained.

(2) Preparation of nano-gold cluster modified electrode

The glassy carbon electrode was sequentially polished and polished with 1.0 μm, 0.3 μm and 0.05 μm Al ₂ O ₃ powder to a smooth mirror surface, and then ultrasonically cleaned in HNO ₃ solution, absolute ethanol, and deionized water for 3 minutes, N ₂ Blow dry. 5 μL of nano-gold cluster aqueous solution was added dropwise on the surface of the treated glassy carbon electrode, and dried at room temperature to obtain nano-gold cluster modified glassy carbon electrode.

(3) Preparation of nano-gold cluster probes

(1) Electrochemical reduction method: a three-electrode system is used for reduction, the nano-gold cluster modified glassy carbon electrode is used as the working electrode, the platinum wire electrode is used as the counter electrode, and Ag/AgCl is used as the reference electrode. The above electrodes are inserted into the buffer solution. Negative potential voltage (in the range of -1.4 V to -2 V) was applied, and the constant potential reduction treatment was carried out for 5 min to 1 h to obtain the electrochemiluminescence nano-gold cluster probe.

(2) Chemical reduction method: soak the nano-gold cluster modified glassy carbon electrode in 0.1-1 mol/L sodium borohydride solution for 5-30 minutes (preferably react in 0.1 mol/L sodium borohydride solution for 5 minutes) to obtain chemical reduction Nano-gold cluster probe modified electrode prepared by the method.

(4) Modification method of manganese dioxide nanomaterials

The electrochemical deposition method is used to modify manganese dioxide nanomaterials, and the specific steps are as follows: a three-electrode system is used, and the electrode modified by the reduction method is used as the working electrode, the platinum wire electrode is used as the counter electrode, and the Ag/AgCl is used as the reference electrode. It method, in KMnO ₄ -H ₂ SO ₄ (1:1 V/V) solution, electrochemically deposit manganese dioxide, the deposition potential is -0.1 V~-0.5 V, the deposition time is 100 s~600 s, preferably -0.2 V, 300 s, then rinsed with double distilled water, and blown dry with nitrogen for later use.

(5) Electrochemiluminescence signal acquisition method

The electrodes were polished, then ultrasonically cleaned in HNO ₃ solution, absolute ethanol and deionized water, and dried in N ₂ . A three-electrode system was used for the test. The modified glassy carbon electrode was used as the working electrode, the platinum wire electrode was used as the counter electrode, and the Ag/AgCl was used as the reference electrode. The above electrodes were inserted into the buffer solution containing co-reactants. The step pulse method was adopted, the initial potential was 0 V, the pulse time was 10 s, the end potential was -2 V, and the pulse time was 1 s. The high voltage of the photomultiplier tube was set at 600 V to 800 V to detect the electrochemiluminescent signal generated on the surface of the working electrode.

The electrodes are glassy carbon electrodes, screen printing electrodes or ITO electrodes and the like. The above co-reactant is persulfate ion, the concentration range is 0.01-1 mol/L, preferably 0.1 mol/L. The buffer solution is a phosphate buffer solution or a Tris-HCl buffer solution, and the electrolyte added in the buffer solution is KCl or KNO ₃ , with a concentration of 0.01~1 mol/L, preferably 0.1 mol/L.

(6) Determination of alkaline phosphatase

Add different concentrations of alkaline phosphatase to 37.5 µL 4-10 mM 2-phosphate-L-ascorbic acid trisodium salt solution, mix well, and then add 300 µL Tris-HCl buffer solution to modify the manganese dioxide/nano-gold clusters The electrode was immersed in the above reaction solution to react for 4-10 min, rinsed with double distilled water after taking it out, and blown dry with _N2 . The electrochemiluminescence signal generated on the surface of the working electrode was collected, and the standard curve was drawn by plotting the concentration of ascorbic acid with the electrochemiluminescence signal.

Compared with prior art, the beneficial effect of the present invention is:

The invention uses high quantum yield nano-gold cluster probes as electrochemiluminescence materials, and nano-manganese dioxide as electrochemiluminescence quenchers, and selectively hydrolyzes 2-phosphate-L-trisodium ascorbate based on alkaline phosphatase The redox reaction between the ascorbic acid produced by the salt and the manganese dioxide recovers the electrochemiluminescence signal, and realizes the detection of the alkaline phosphatase content. The invention has the advantages of simple operation, low cost, high sensitivity, good stability, good accuracy, wide linear range (1.0×10 ^-9 ~1.0×10 ^-2 mol/L) and low detection limit for the detection of alkaline phosphatase (2.56×10 ^-10 mol/L), so it has a good market value.

Description of drawings

Fig. 1 is the electrochemiluminescence-time graph of the nano-gold cluster probe modified glassy carbon electrode.

Fig. 2 is the electrochemiluminescence-time curve of the manganese dioxide/nano-gold cluster probe modified glassy carbon electrode.

Fig. 3 is the electrochemiluminescence-time graph of the manganese dioxide/nano-gold cluster probe modified electrode immersed in the reaction solution of alkaline phosphatase and 2-phospho-L-ascorbic acid trisodium salt.

Fig. 4 is an optimization diagram of the concentration of 2-phospho-L-ascorbic acid trisodium salt.

Figure 5 is a graph showing the linear relationship between the change in the intensity of electrochemiluminescence and the logarithm value of the concentration of alkaline phosphatase.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

Example 1

Add 0.6 mL of 0.5 mol/L sodium hydroxide and 0.4 mL of 20 mg/mL chloroauric acid solution to 4 mL of 0.08 mol/L N-acetyl-L-cysteine solution, mix After uniformity, they were incubated in a constant temperature water tank at 37 °C for 3 h. After the reaction, the reaction solution was purified by dialysis with a dialysis bag with a molecular cut-off of 3500 to obtain an aqueous solution of N-acetyl-L-cysteine-nano-gold clusters, which was freeze-dried to obtain N-acetyl-L-cysteine -Nano gold cluster powder.

Example 2

A glassy carbon electrode with a diameter of 3 mm was polished with 1.0 μm, 0.3 μm and 0.05 μm Al ₂ O ₃ powder in sequence, and polished to a smooth mirror surface, and then put in HNO ₃ solution, absolute ethanol, and deionized water for ultrasonic cleaning for 3 minutes , blow dry with N ₂ . Take 5 μL of the N-acetyl-L-cysteine-nano-gold cluster solution prepared in Example 1 and drop it on the surface of the treated glassy carbon electrode, and dry it at room temperature to obtain N-acetyl-L-cysteine-nano Gold cluster modified glassy carbon electrodes. The N-acetyl-L-cysteine-nano-gold cluster modified glassy carbon electrode was soaked in 0.1 mol/L sodium borohydride solution for 5 min to obtain the nano-gold cluster probe-modified electrode. The above nano-gold cluster probe modified electrode was used as the working electrode, the platinum wire was used as the counter electrode, and the Ag/AgCl electrode was used as the reference electrode, and a 0.1 mol/L pH 7.4 solution containing 0.1 mol/L potassium persulfate and 0.1 mol/L KCl was inserted. in phosphate buffered saline solution. The step pulse method was adopted, the initial potential was 0 V, the pulse time was 10 s, the end potential was -2 V, and the pulse time was 1 s. The high voltage of the photomultiplier tube was set to 750 V, and the electrochemiluminescence signal generated on the surface of the working electrode was detected to obtain the electrochemiluminescence signal (see Figure 1).

Example 3

A glassy carbon electrode with a diameter of 3 mm was sequentially polished with 1.0 μm, 0.3 μm and 0.05 μm Al ₂ O ₃ powder, polished to a smooth mirror surface, and then placed in HNO ₃ solution, absolute ethanol, and deionized water for ultrasonic cleaning for 3 minutes, blow dry with N ₂ . Take 5 μL of the N-acetyl-L-cysteine-protected nano-gold cluster solution prepared in Example 1 and drop it on the surface of the treated glassy carbon electrode, dry it at room temperature, and further soak the electrode in 0.1 mol/L boron React in sodium hydride solution for 5 minutes to obtain nano-gold cluster probe-modified glassy carbon electrode. A three-electrode system was adopted, with a nano-gold cluster probe-modified glassy carbon electrode as the working electrode, a platinum wire electrode as the counter electrode, and Ag/AgCl as the reference electrode. Chronoamperometry was used in KMnO ₄ -H ₂ SO ₄ (1: 1 V/V) solution, manganese dioxide was electrodeposited, the deposition potential was -0.2 V, and the deposition time was 300 s. The obtained electrode was manganese dioxide/nano-gold cluster modified glassy carbon electrode, and then rinsed with double distilled water Clean and blow dry gently with N ₂ gas for later use. Insert the above electrodes into 0.1 mol/L pH 7.4 phosphate buffer solution containing 0.1 mol/L potassium persulfate and 0.1 mol/L KCl. The step pulse method was adopted, the initial potential was 0 V, the pulse time was 10 s, the end potential was -2 V, and the pulse time was 1 s. The high voltage of the photomultiplier tube was set to 750 V, and the electrochemiluminescence signal generated on the surface of the working electrode was detected to obtain the electrochemiluminescence signal (see Figure 2).

Example 4

Add 400 U/L alkaline phosphatase to 37.5 µL 4 mM 2-Phospho-L-ascorbic acid trisodium salt solution (2-Phospho-L-ascorbic acid trisodium salt, purchased from Sigma-Aldrich Company), mix well, and then add 300 µL Tris-HCl buffer solution, and then the manganese dioxide/nano-gold cluster modified electrode was immersed in the above reaction solution for 4 min. The above-mentioned manganese dioxide/nano-gold cluster probe-modified electrode was used as the working electrode, the platinum wire was used as the counter electrode, and the Ag/AgCl electrode was used as the reference electrode, and 0.1 mol/L potassium persulfate and 0.1 mol/L KCl were inserted into the electrode. /L, pH 8.0 phosphate buffer solution. The step pulse method was adopted, the initial potential was 0 V, the pulse time was 10 s, the end potential was -2 V, and the pulse time was 1 s. The high voltage of the photomultiplier tube was set to 750 V, and after 4 min of reaction, it was taken out, rinsed with double distilled water, and dried with N ₂ . Detect the electrochemiluminescent signal produced on the surface of the working electrode, and the obtained electrochemiluminescent signal is significantly larger than the electrochemiluminescent signal in the reaction solution of alkaline phosphatase and 2-phospho-L-ascorbic acid trisodium salt solution ( See Figure 3).

Example 5

Add 400 U/L alkaline phosphatase to 37.5 μL different concentrations of 2-phosphate-L-ascorbic acid trisodium salt solution, mix well, then add 300 μL Tris-HCl buffer solution, and then the manganese dioxide prepared in Example 3 /Nano-gold cluster modified electrode was immersed in the above reaction solution for 4 min and reacted for 4 min. After taking it out, it was rinsed with double distilled water and dried with N ₂ . The above air-dried manganese dioxide/nano-gold cluster probe-modified electrode was used as the working electrode, the platinum wire was used as the counter electrode, and the Ag/AgCl electrode was used as the reference electrode, and the insert containing 0.1 mol/L potassium persulfate and 0.1 mol/L KCl 0.1 mol/L, pH 8.0 phosphate buffer solution. The step pulse method was adopted, the initial potential was 0 V, the pulse time was 10 s, the end potential was -2 V, and the pulse time was 1 s. The high voltage of the photomultiplier tube was set to 750 V, and after 4 minutes of reaction, the concentration of 2-phospho-L-ascorbic acid trisodium salt solution was detected as 0, 1 mM, 2 mM, 3 mM, 4 mM, 6 mM, 8 mM and 10 mM When the electrochemiluminescence signal generated on the surface of the working electrode was used, the optimal concentration of 2-phospho-L-ascorbic acid trisodium salt solution was 4 mM (see Figure 4).

Example 6

Add alkaline phosphatase of different concentrations to 37.5 µL 4 mM 2-phospho-L-ascorbic acid trisodium salt solution, mix well, then add 300 µL Tris-HCl buffer solution, and then the manganese dioxide prepared in Example 3 /Nano-gold cluster modified electrode was immersed in the above reaction solution for 4 min and reacted for 4 min. After taking it out, it was rinsed with double distilled water and dried with N ₂ . The above air-dried manganese dioxide/nano-gold cluster probe-modified electrode was used as the working electrode, the platinum wire was used as the counter electrode, and the Ag/AgCl electrode was used as the reference electrode, and the insert containing 0.1 mol/L potassium persulfate and 0.1 mol/L KCl 0.1 mol/L, pH 8.0 phosphate buffer solution. The step pulse method was adopted, the initial potential was 0 V, the pulse time was 10 s, the end potential was -2 V, and the pulse time was 1 s. The high voltage of the photomultiplier tube was set to 750 V, and after ⁴ minutes of reaction, the electrochemiluminescence signals generated on the surface of the working electrode were detected under the conditions of different concentrations of alkaline phosphatase. The logarithmic value of glutathione concentration in the range of ×10 ² U/L has a good linear relationship with the intensity of electrochemiluminescence, and the detection limit is 1.5×10 ^-5 U/L (see Figure 5).

Example 7

Take 2 µL of fresh serum sample, add 20 mM Tris-HCl buffer to dilute to 20 mL, add the diluted serum sample to Tris-HCl buffer containing 2-phospho-L-ascorbic acid trisodium salt, and then implement The manganese dioxide/nano-gold cluster modified electrode prepared in Example 3 was soaked in the above solution for 4 minutes, rinsed with double distilled water after taking it out, and dried with N ₂ . The above air-dried manganese dioxide/nano-gold cluster probe-modified electrode was used as the working electrode, the platinum wire was used as the counter electrode, and the Ag/AgCl electrode was used as the reference electrode, and the insert containing 0.1 mol/L potassium persulfate and 0.1 mol/L KCl 0.1 mol/L, pH 8.0 phosphate buffer solution. The step pulse method was adopted, the initial potential was 0 V, the pulse time was 10 s, the end potential was -2 V, and the pulse time was 1 s. The high voltage of the photomultiplier tube was set to 750 V, and after 4 min of reaction, the electrochemiluminescence signals generated on the surface of each working electrode in different sample solutions were detected respectively, and quantified by the standard curve to obtain the content of alkaline phosphatase in the sample, and used The international alkaline phosphatase detection standard method PNPP azo method was used for detection, and then statistically compared with the detection results of this experimental method (Table 1). There is no significant difference between this method and PNPP method, therefore, this experimental method is suitable for the detection of actual samples.

Table 1 shows the detection results of alkaline phosphatase in actual samples.

The above descriptions are only typical embodiments of the present invention, and are not intended to limit the present invention. Any modifications made within the spirit and principles of the present invention, equivalent replacements and improvements, etc., should be included in the protection scope of the present invention within.

Claims (8)

1. a kind of alkaline phosphatase assay method based on nanogold cluster electrogenerated chemiluminescence probe, it is characterized in that：First in electricity Pole surface modified nano gold cluster prepares high quantum production rate nanogold cluster electrogenerated chemiluminescence probe by reduction method, and further In electrode face finish nano material of manganese dioxide, as electrogenerated chemiluminescence quencher；2- is added in alkaline phosphatase In phosphoric acid-L-AA trisodium-salt solution, alkaline phosphatase selective hydrolysis 2- phosphoric acid-L-AA trisodium salt generates anti- Bad hematic acid, and then modified electrode is immersed in above-mentioned reaction solution, it is anti-that redox occurs for ascorbic acid and the manganese dioxide of generation It answers；The electrochemiluminescence signal for acquiring modified electrode realizes alkaline phosphatase according to the change of electrochemiluminescence signal Measurement.

2. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1 Method, it is characterized in that the reduction method, which prepares high quantum production rate nanogold cluster electrogenerated chemiluminescence probe, uses following electrochemistry also Former method or chemical reduction method preparation：

（1）Electrochemical reducing：Using three-electrode system, using nanogold cluster modified glassy carbon electrode as working electrode, platinum electrode For to electrode, Ag/AgCl is reference electrode, 0.1 mol/L, pH 7.4 of above-mentioned electrode insertion in phosphate buffer solution, is applied Add negative potential voltage within the scope of -1.4 V of V ~ -2, carries out constant potential reduction treatment 5 min ~ 1 h, obtain electrochemical luminescence nanometer Golden aggregate probe；

（2）Chemical reduction method：Nanogold cluster modified glassy carbon electrode is immersed in 0.1 ~ 1 mol/L sodium borohydride solution reaction 5 The min of min ~ 30 obtains the nanogold aggregate probe modified electrode of chemical reduction method preparation.

3. a kind of according to claim 1, alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe described in 2 Method, it is characterized in that the nanogold cluster is functional modification nanogold cluster, using N- acetylation-L-cysteine-nanogold cluster Or bovine serum albumin(BSA)-nanogold cluster.

4. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1 Method, it is characterized in that the method in electrode face finish nano material of manganese dioxide is electrochemical deposition method：Using three electrodes System, using reduction method processing nanogold cluster modified electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electricity Pole, using I-t method, 1:The KMnO of 1 V/V₄-H₂SO₄In solution, electrochemical deposition manganese dioxide, current potential be -0.1 V ~ - 0.5 V, sedimentation time are the s of 100 s ~ 600, and cleaning is dried with nitrogen.

5. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1 Method, it is characterized in that the electrochemiluminescence signal method of the acquisition modified electrode is as follows：It is tested using three-electrode system, Using modified electrode as working electrode, platinum electrode is to electrode, and Ag/AgCl is reference electrode, and buffer solution is phosphate-buffered Solution or Tris-HCl buffer solution, electrolyte used are KCl or KNO₃；The insertion of above-mentioned electrode is total to containing over cure acid ion In the buffer solution of reactant, electrochemical luminescence test is carried out using step pulse method, initial potential is 0 V, and the burst length is 10 s, termination current potential are -2 V, and the burst length is 1 s, and photomultiplier tube high pressure is set as 600 ~ 800 V, detects electroluminescent chemistry Luminous radiation signal.

6. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1 Method, it is characterized in that the pH that redox reaction occurs for the ascorbic acid of the generation and manganese dioxide is 8.0, the reaction time 4 min。

7. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 5 Method, it is characterized in that the phosphate buffer solution or Tris-HCl buffer solution of the electrochemiluminescence signal of acquisition modified electrode PH value is 7.4, and persulfuric acid ion concentration is 0.1 mol/L.

8. a kind of alkaline phosphatase assay side based on nanogold cluster electrogenerated chemiluminescence probe according to claim 1 Method, it is characterized in that the logarithm of electrogenerated chemiluminescence Strength Changes value and ascorbic acid concentrations is 1.0 × 10^-4~2.0×10² U/ It is in good linear relationship in the range of L, detection is limited to 1.5 × 10^-5 U/L。