阅读说明：本技术 一种检测唾液酸化糖链抗原kl-6铱纳米点电化学发光传感器的制备及应用 (Preparation and application of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots ) 是由 杜宇 任祥 吴丹 刘雪静 范大伟 魏琴 鞠熀先 于 2020-12-27 设计创作，主要内容包括：本发明涉及一种检测唾液酸化糖链抗原KL-6铱纳米点电化学发光传感器的制备及应用,其特征在于,制备一种水溶性好、生物相容性好、电化学发光效率高的铱纳米点,以抗体标记的铱纳米点溶液作为信号探针,研制一种无标记型电化学发光免疫传感器,用于间质性肺疾病标志物唾液酸化糖链抗原KL-6的快速、灵敏检测,检出限为45 fg/mL,线性范围为100 fg/mL-50 ng/mL,具有特异性强、重现性好、信号稳定等优点。(The invention relates to preparation and application of an iridium nanodot electrochemiluminescence sensor for detecting sialylation sugar chain antigen KL-6, which is characterized in that an iridium nanodot with good water solubility, good biocompatibility and high electrochemiluminescence efficiency is prepared, an iridium nanodot solution marked by an antibody is used as a signal probe to develop a label-free electrochemiluminescence immunosensor, and the label-free electrochemiluminescence immunosensor is used for quickly and sensitively detecting sialylation sugar chain antigen KL-6 serving as an interstitial lung disease marker, has the detection limit of 45 fg/mL and the linear range of 100 fg/mL-50 ng/mL, and has the advantages of strong specificity, good reproducibility, stable signal and the like.)

1. The preparation and application of the electrochemical luminescence sensor for detecting the sialylated sugar chain antigen KL-6 iridium nanodots are characterized by comprising the following steps of:

(1) preparation of iridium nanodot solution

Dissolving iridium complex solid with the mass of 10-20 mg in 1-4 mL tetrahydrofuran at room temperature to prepare a solution with the mass concentration of 5-10 mg/mL; uniformly shaking a sodium hydroxide solution with the molar concentration of 0.1-0.3 mol/L and a apoferritin solution with the mass concentration of 0.5-1.0 mg/mL according to a certain volume ratio, and continuously stirring for 2-4 h at room temperature to completely dissociate an apoferritin structural unit into free subunits; then, 0.5-1.5 mL of iridium complex solution with the mass concentration of 5-10 mg/mL is dropwise added into the mixed solution, and after the mixture is continuously stirred for 2-4 hours, 0.1-0.3 mL of hydrochloric acid solution with the molar concentration of 0.1-0.3 mol/L is rapidly added into the mixed solution to neutralize the solution; after the mixed solution is stood for 2-4 hours, separating and purifying the product by using an ultrafiltration centrifugal tube, and dispersing the finally prepared iridium nanodots into 5-7 mL of phosphate buffer solution with the pH value of 7.4 for later use;

(2) preparation of iridium nanodot solution labeled by antibody

Adding 50-70 mu L of glutaraldehyde with the mass fraction of 30% and 500-700 mu L of antibody standard solution (10 mu g/mL) into 1 mL of iridium nanodot solution, incubating for 12-24 h at 4 ℃, performing centrifugal separation to obtain an antibody-labeled iridium nanodot solution, and storing at 4 ℃ for later use;

(3) preparation steps of iridium nanodot electrochemiluminescence sensor

a. Polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;

b. dripping 6 mu L of antibody-labeled iridium nanodot solution with the concentration of 2-3 mg/mL on the surface of the glassy carbon electrode, and airing at 37 ℃;

c. dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1-3% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;

d. dripping 6 mu L of sialylated sugar chain antigen KL-6 solution of the interstitial lung disease marker with the concentration of 10 mu g/mL or unknown concentration, incubating at 37 ℃ for 1 h, washing the surface of the electrode by phosphate buffer solution PBS with pH of 7.4, and finishing the preparation of the immunosensor;

(4) an iridium nanodot electrochemiluminescence immunosensor is used for detecting sialylation carbohydrate chain antigen KL-6 of interstitial lung disease markers.

2. The preparation and application of the electrochemiluminescence sensor for detecting sialylated oligosaccharide antigen KL-6 iridium nanodots as claimed in claim 1, wherein the iridium complex is bis (2-phenylpyridine) acetylacetone iridium Ir (ppy)₂(acac)。

3. The preparation and application of the electrochemiluminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots according to claim 1, wherein the apoferritin is biotin-labeled equine spleen apoferritin.

4. The preparation and application of the sialylation carbohydrate chain antigen KL-6 iridium nanodot electrochemiluminescence sensor according to claim 1, wherein the volume ratio of the solution of sodium hydroxide to the solution of apoferritin is 1: 100.

5. The iridium nanodot electrochemiluminescence immunosensor prepared by the preparation method according to claim 1 is used for detecting sialylated carbohydrate chain antigen KL-6 as a marker of interstitial lung disease, and is characterized by comprising the following operation steps:

(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemical luminometer is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-1.0V, and the scanning rate is set to be 0.1V/s;

(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out an electrochemiluminescence test in 10 mL of phosphate buffer solution containing 25-45 mmol/L triethylamine to obtain corresponding electrochemiluminescence signal intensity when the sialylated carbohydrate chain antigen KL-6 with different concentrations is incubated, and drawing a working curve (the detection limit is 45 fg/mL, and the linear range is 100 fg/mL-50 ng/mL);

(3) and testing the sensor for hatching the actual sample of the sialylated sugar chain with unknown concentration, and calculating according to the working curve to obtain the concentration of the sialylated sugar chain in the actual sample.

Technical Field

The invention belongs to the field of nano materials and the technical field of biosensing.

Background

As a research hotspot which is raised by the interdigitation of various subjects such as biology, chemistry, medicine, electronic technology and the like, the electrochemiluminescence immunoassay is the organic combination of the electrochemiluminescence technology and an immunoassay method, and the prepared immunosensor has the advantages of low cost, good selectivity, high sensitivity, high analysis speed, easiness in automation, miniaturization, integration and the like, and is widely applied to the fields of disease marker analysis, food safety analysis, environmental pollution analysis and the like.

The interstitial lung disease has the characteristics of wide disease population, strong hiding property, long disease course, complex disease mechanism, great difficulty in diagnosis and treatment and the like. Such diseases, which lead to damage and corresponding dysfunction of the lung organs, have become a hot and difficult problem of clinical interest in recent years. Bronchoscopy and surgical lung biopsy are not suitable for repeated examination in a short period, and lung function examination requires high patient matching degree and many influencing factors, so that the bronchoscopy and surgical lung biopsy are not suitable for routine detection of severe patients. Therefore, it is important to find a noninvasive biological index of serum that can be used for ILD diagnosis and disease monitoring. The sialylated sugar chain antigen KL-6 is used as a brand new disease marker for detecting interstitial lung diseases, has the characteristics of high specificity and high sensitivity, and can be used for auxiliary diagnosis of the interstitial lung diseases. However, immunoassay methods related thereto have been reported only rarely. Therefore, based on the superiority of the electrochemiluminescence immunoassay, a simple, rapid and accurate electrochemiluminescence immunosensor is developed to be used for measuring the sialylated carbohydrate chain antigen KL-6, and the electrochemiluminescence immunosensor has important research significance and clinical application value.

In recent years, the design and synthesis of phosphorescent transition metal complexes have become a focus of research. Iridium complexes as the largest class of complexes in the family of organic electroluminescent devicesThe development of the parts has wide application. With extremely high electrochemical luminescence efficiency, with bis (2-phenylpyridine) acetylacetone iridium Ir (ppy)₂Iridium complexes represented by (acac) have begun to attract attention in the field of bioanalysis. However, Ir (ppy)₂(acac) has poor water solubility, easy agglomeration and poor biocompatibility, and greatly limits the application of the (acac) in water-phase immunoassay.

Disclosure of Invention

(1) One of the purposes of the invention is to overcome the defects, prepare an iridium nanodot with good water solubility, good biocompatibility and high electrochemical luminescence efficiency, and develop a label-free electrochemical luminescence immunosensor by taking an iridium nanodot solution labeled by an antibody as a signal probe;

(2) the invention also aims to provide the application of the immunosensor, the immunosensor is used for detecting the concentration of the sialylated sugar chain antigen KL-6 of the interstitial lung disease marker, the signal response of the sensor is rapid, the sensitivity is high, the detection time is greatly shortened, the manpower, material resources and financial resources are saved, the detection limit is 45 fg/mL, and the linear range is 100 fg/mL-50 ng/mL.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

preparation and application of an electrochemiluminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots, which comprises the following steps:

1. preparation of iridium nanodot solution

Dissolving iridium complex solid with the mass of 10-20 mg in 1-4 mL tetrahydrofuran at room temperature to prepare a solution with the mass concentration of 5-10 mg/mL; uniformly shaking a sodium hydroxide solution with the molar concentration of 0.1-0.3 mol/L and a apoferritin solution with the mass concentration of 0.5-1.0 mg/mL according to a certain volume ratio, and continuously stirring for 2-4 h at room temperature to completely dissociate an apoferritin structural unit into free subunits; then, 0.5-1.5 mL of iridium complex solution with the mass concentration of 5-10 mg/mL is dropwise added into the mixed solution, and after the mixture is continuously stirred for 2-4 hours, 0.1-0.3 mL of hydrochloric acid solution with the molar concentration of 0.1-0.3 mol/L is rapidly added into the mixed solution to neutralize the solution; after the mixed solution is stood for 2-4 hours, separating and purifying the product by using an ultrafiltration centrifugal tube, and dispersing the finally prepared iridium nanodots into 5-7 mL of phosphate buffer solution with the pH value of 7.4 for later use;

the iridium complex is bis (2-phenylpyridine) acetylacetone iridium Ir (ppy)₂(acac)；

The apoferritin is biotin-labeled equine spleen apoferritin;

the volume ratio of the solution of the sodium hydroxide to the solution of the apoferritin is 1: 100.

2. Preparation of iridium nanodot solution labeled by antibody

Adding 50-70 mu L of glutaraldehyde with the mass fraction of 30% and 500-700 mu L of antibody standard solution (10 mu g/mL) into 1 mL of iridium nanodot solution, incubating at 4 ℃ for 12-24 h, performing centrifugal separation to obtain an antibody-labeled iridium nanodot solution, and storing at 4 ℃ for later use;

3. preparation of electrochemical luminescence sensor for detecting sialylation carbohydrate chain antigen KL-6 iridium nanodot

a. Polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;

b. dripping 6 mu L of antibody-labeled iridium nanodot solution with the concentration of 2-3 mg/mL on the surface of the glassy carbon electrode, and airing at 37 ℃;

c. dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1-3% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;

d. dripping 6 mu L of sialylated sugar chain antigen KL-6 solution of the interstitial lung disease marker with the concentration of 10 mu g/mL or unknown concentration, incubating at 37 ℃ for 1 h, washing the surface of the electrode by phosphate buffer solution PBS with pH of 7.4, and finishing construction of the immunosensor;

4. an iridium nanodot electrochemiluminescence immunosensor is used for detecting sialylation carbohydrate chain antigen KL-6 as a marker of interstitial lung disease, and the operation steps are as follows:

(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemical luminometer is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-1.0V, and the scanning rate is set to be 0.1V/s;

(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out an electrochemiluminescence test in 10 mL of phosphate buffer solution containing 25-45 mmol/L triethylamine to obtain corresponding electrochemiluminescence signal intensity when the sialylated carbohydrate chain antigen KL-6 with different concentrations is incubated, and drawing a working curve (the detection limit is 45 fg/mL, and the linear range is 100 fg/mL-50 ng/mL);

(3) and testing the sensor for hatching the actual sample of the sialylated sugar chain with unknown concentration, and calculating according to the working curve to obtain the concentration of the sialylated sugar chain in the actual sample.

Advantageous results of the invention

(1) Firstly, a method for efficiently packaging iridium complex molecules in the interior of apoferritin is provided, so that the prepared iridium nanodot has good water solubility and high electrochemical luminescence efficiency, and the problems of poor water solubility and poor biocompatibility of the iridium complex in a water phase are solved;

(2) by taking the iridium nanodot solution marked by the antibody as a signal probe, developing a label-free electrochemical luminescence immunosensor for detecting sialylated sugar chain antigen KL-6 as a marker of interstitial lung disease;

(3) the blank of the prior sialylated carbohydrate chain antigen KL-6 electrochemiluminescence detection method is made up, in the actual sample detection, the detection limit of the immunosensor is as low as 45 fg/mL, and the linear range is as wide as 100 fg/mL-50 ng/mL;

(4) the developed immunosensor has the advantages of simplicity in preparation, easiness in operation, rapidness in signal response, good reproducibility, environmental friendliness, no pollution and the like, has strong specificity on sialylated carbohydrate chain antigen KL-6 in serum, and has great potential application in early diagnosis of interstitial lung diseases.

Detailed Description

The invention will now be further illustrated by reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.

Embodiment 1. preparation and application of an iridium nanodot electrochemiluminescence sensor for detecting sialylation carbohydrate chain antigen KL-6, an iridium nanodot solution comprises the following steps:

at room temperature, 10 mg by mass of bis (2-phenylpyridine) acetylacetonatoiridium Ir (ppy)₂(acac) the solid was dissolved in 1 mL tetrahydrofuran to prepare a solution with a mass concentration of 10 mg/mL; uniformly shaking a sodium hydroxide solution with the molar concentration of 0.1 mol/L and a apoferritin solution with the mass concentration of 0.5 mg/mL according to the volume ratio of 1:100, and continuously stirring for 2 hours at room temperature to completely dissociate an apoferritin structural unit into free subunits; then, 0.5 mL of iridium complex solution with the mass concentration of 10 mg/mL is dropwise added into the mixed solution, after the mixture is continuously stirred for 2 hours, 0.1 mL of hydrochloric acid solution with the molar concentration of 0.1 mol/L is rapidly added into the mixed solution to neutralize the solution; and (3) after the mixed solution is stood for 2 hours, separating and purifying the product by using an ultrafiltration centrifugal tube, and dispersing the finally prepared iridium nanodots into 5 mL of phosphate buffer solution with the pH value of 7.4 for later use.

Embodiment 2. preparation and application of an iridium nanodot electrochemiluminescence sensor for detecting sialylation sugar chain antigen KL-6, the preparation steps of an iridium nanodot solution are as follows:

bis (2-phenylpyridine) acetylacetonatoiridium Ir (ppy) with a mass of 15 mg at room temperature₂(acac) was dissolved in 2 mL of tetrahydrofuran to prepare a solution with a mass concentration of 7.5 mg/mL; uniformly shaking a sodium hydroxide solution with the molar concentration of 0.2 mol/L and a apoferritin solution with the mass concentration of 0.7 mg/mL according to the volume ratio of 1:100, and continuously stirring for 3 hours at room temperature to completely dissociate an apoferritin structural unit into free subunits; then, 1 mL of iridium complex solution with the mass concentration of 7.5 mg/mL is dropwise added into the mixed solution, after the mixture is continuously stirred for 3 hours, 0.2 mL of hydrochloric acid solution with the molar concentration of 0.2 mol/L is rapidly added into the mixed solution to neutralize the solution; and after standing the mixed solution for 3 hours, separating and purifying the product by using an ultrafiltration centrifugal tube, and dispersing the finally prepared iridium nanodots into 6 mL of phosphate buffer solution with the pH value of 7.4 for later use.

Embodiment 3. preparation and application of an iridium nanodot electrochemiluminescence sensor for detecting sialylation sugar chain antigen KL-6, the preparation steps of an iridium nanodot solution are as follows:

at room temperature, 20 mg by mass of bis (2-phenylpyridine) acetylacetonatoiridium Ir (ppy)₂(acac) was dissolved in 4 mL of tetrahydrofuran to prepare a solution with a mass concentration of 5 mg/mL; uniformly shaking a sodium hydroxide solution with the molar concentration of 0.3 mol/L and a apoferritin solution with the mass concentration of 5 mg/mL according to the volume ratio of 1:100, and continuously stirring for 4 hours at room temperature to completely dissociate an apoferritin structural unit into free subunits; then, 1.5 mL of iridium complex solution with the mass concentration of 5 mg/mL is gradually dripped into the solution, and after the solution is continuously stirred for 4 hours, 0.3 mL of hydrochloric acid solution with the molar concentration of 0.3 mol/L is rapidly added into the mixed solution to neutralize the solution; and after standing the mixed solution for 4 hours, separating and purifying the product by using an ultrafiltration centrifugal tube, and dispersing the finally prepared iridium nanodots into 7 mL of phosphate buffer solution with the pH value of 7.4 for later use.

Embodiment 4. preparation and application of an iridium nanodot electrochemiluminescence sensor for detecting sialylation sugar chain antigen KL-6, an iridium nanodot solution marked by an antibody is prepared according to the following steps:

taking 1 mL of the prepared iridium nanodot solution, adding 50 mu L of glutaraldehyde with the mass fraction of 30% as a cross-linking agent, then adding 500 mu L of antibody standard solution with the concentration of 10 mu g/mL, incubating at 4 ℃ for 12 h, obtaining an antibody-labeled iridium nanodot solution through centrifugal separation, and storing at 4 ℃ for later use.

Embodiment 5. preparation and application of an iridium nanodot electrochemiluminescence sensor for detecting sialylation sugar chain antigen KL-6, an iridium nanodot solution marked by an antibody is prepared according to the following steps:

and taking 1 mL of the prepared iridium nanodot solution, adding 60 mu L of glutaraldehyde with the mass fraction of 30% as a cross-linking agent, then adding 600 mu L of antibody standard solution with the concentration of 10 mu g/mL, incubating for 18 h at 4 ℃, obtaining an antibody-labeled iridium nanodot solution through centrifugal separation, and storing at 4 ℃ for later use.

Embodiment 6. preparation and application of an electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots, an iridium nanodot solution marked by an antibody is prepared according to the following steps:

taking 1 mL of the prepared iridium nanodot solution, adding 70 mu L of glutaraldehyde with the mass fraction of 30% as a cross-linking agent, then adding 700 mu L of antibody standard solution with the concentration of 10 mu g/mL, incubating for 24 h at 4 ℃, obtaining an antibody-labeled iridium nanodot solution through centrifugal separation, and storing at 4 ℃ for later use.

Embodiment 7. preparation and application of an electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots, the construction steps of the immunosensor are as follows:

a. polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;

b. dripping 6 mu L of antibody-labeled iridium nanodot solution with the concentration of 2 mg/mL on the surface of the glassy carbon electrode, and airing the glassy carbon electrode at 37 ℃;

c. dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1% to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;

d. 6 mu L of interstitial lung disease marker sialylated sugar chain antigen KL-6 solution with the concentration of 10 mu g/mL or unknown concentration is dripped, the mixture is incubated for 1 h at 37 ℃, the surface of an electrode is washed by phosphate buffer solution PBS with the pH of 7.4, and the construction of the immunosensor is finished.

Embodiment 8. preparation and application of an electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots, the construction steps of the immunosensor are as follows:

a. polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;

b. dripping 6 mu L of antibody-labeled iridium nanodot solution with the concentration of 3 mg/mL on the surface of the glassy carbon electrode, and airing the glassy carbon electrode at 37 ℃;

c. dropwise adding 3 mu L of 2% bovine serum albumin solution to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution PBS (pH 7.4), and airing at 4 ℃;

d. 6 mu L of interstitial lung disease marker sialylated sugar chain antigen KL-6 solution with the concentration of 10 mu g/mL or unknown concentration is dripped, the mixture is incubated for 1 h at 37 ℃, the surface of an electrode is washed by phosphate buffer solution PBS with the pH of 7.4, and the construction of the immunosensor is finished.

Embodiment 9. preparation and application of an electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots, the construction steps of the immunosensor are as follows:

a. polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;

b. dripping 6 mu L of antibody-labeled iridium nanodot solution with the concentration of 4 mg/mL on the surface of the glassy carbon electrode, and airing the glassy carbon electrode at 37 ℃;

c. dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 3% to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;

d. 6 mu L of interstitial lung disease marker sialylated sugar chain antigen KL-6 solution with the concentration of 10 mu g/mL or unknown concentration is dripped, the mixture is incubated for 1 h at 37 ℃, the surface of an electrode is washed by phosphate buffer solution PBS with the pH of 7.4, and the construction of the immunosensor is finished.

Example 10 an iridium nanodot electrochemiluminescence immunosensor is used for sialylation carbohydrate chain antigen KL-6 as a marker of interstitial lung disease, and the operation steps are as follows:

(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemical luminometer is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-1.0V, and the scanning rate is set to be 0.1V/s;

(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out an electrochemiluminescence test in 10 mL of phosphate buffer solution containing 25 mmol/L triethylamine to obtain corresponding electrochemiluminescence signal intensity when the sialylated carbohydrate chain antigen KL-6 with different concentrations is incubated, drawing a working curve, wherein the detection limit is 45 fg/mL, and the linear range is 100 fg/mL-50 ng/mL;

(3) and testing the sensor for hatching the actual sample of the sialylated sugar chain with unknown concentration, and calculating according to the working curve to obtain the concentration of the sialylated sugar chain in the actual sample.

Example 11 an iridium nanodot electrochemiluminescence immunosensor is used for sialylation carbohydrate chain antigen KL-6 as a marker of interstitial lung disease, and the operation steps are as follows:

(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemical luminometer is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-1.0V, and the scanning rate is set to be 0.1V/s;

(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out an electrochemiluminescence test in 10 mL of phosphate buffer solution containing 35 mmol/L triethylamine to obtain corresponding electrochemiluminescence signal intensity when the sialylated carbohydrate chain antigen KL-6 with different concentrations is incubated, drawing a working curve, wherein the detection limit is 45 fg/mL, and the linear range is 100 fg/mL-50 ng/mL;

(3) and testing the sensor for hatching the actual sample of the sialylated sugar chain with unknown concentration, and calculating according to the working curve to obtain the concentration of the sialylated sugar chain in the actual sample.

Example 12 an iridium nanodot electrochemiluminescence immunosensor is used for sialylation carbohydrate chain antigen KL-6 as a marker of interstitial lung disease, and the operation steps are as follows:

(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemical luminometer is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-1.0V, and the scanning rate is set to be 0.1V/s;

(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared by the method as a working electrode, carrying out an electrochemiluminescence test in 10 mL of phosphate buffer solution containing 45 mmol/L triethylamine to obtain corresponding electrochemiluminescence signal intensity when the sialylated carbohydrate chain antigen KL-6 with different concentrations is incubated, drawing a working curve, wherein the detection limit is 45 fg/mL, and the linear range is 100 fg/mL-50 ng/mL;

(3) and testing the sensor for hatching the actual sample of the sialylated sugar chain with unknown concentration, and calculating according to the working curve to obtain the concentration of the sialylated sugar chain in the actual sample.