阅读说明：本技术 一种检测ⅰ型前胶原氨基端原肽铱纳米晶电化学发光传感器的制备及应用 (Preparation and application of iridium nanocrystalline electrochemical luminescence sensor for detecting I-type procollagen amino-terminal propeptide ) 是由 杜宇 任祥 刘蕾 刘雪静 张诺 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Ⅰ型前胶原氨基端原肽铱纳米晶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晶,以抗体标记的铱纳米晶溶液作为信号探针,研制一种无标记型电化学发光免疫传感器,用于多发性骨髓瘤标志物Ⅰ型前胶原氨基端原肽的快速、灵敏检测,检出限为35 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 a preparation method and application of an iridium nanocrystalline electrochemical luminescence sensor for detecting I-type procollagen amino-terminal propeptide, which is characterized in that an iridium nanocrystalline with good water solubility, good biocompatibility and high electrochemical luminescence efficiency is prepared, an iridium nanocrystalline solution marked by an antibody is used as a signal probe to develop a label-free electrochemical luminescence immunosensor for the rapid and sensitive detection of the I-type procollagen amino-terminal propeptide serving as a multiple myeloma marker, the detection limit is 35 fg/mL, the linear range is 100 fg/mL-50 ng/mL, and the iridium nanocrystalline electrochemical luminescence immunosensor has the advantages of strong specificity, good reproducibility, stable signal and the like.)

1. Preparation and application of an iridium nanocrystalline electrochemical luminescence sensor for detecting I-type procollagen amino-terminal propeptide are characterized by comprising the following steps:

(1) preparation of Iridium nanocrystalline solution

Dissolving iridium complex solid with the mass of 10-20 mg in 1-4 mL tetrahydrofuran at room temperature to prepare iridium complex solution with the mass concentration of 5-10 mg/mL, measuring 0.5-1.5 mL iridium complex solution with the mass concentration of 5-10 mg/mL, dropwise adding the iridium complex solution with the mass concentration of 5-10 mg/mL into 8.4-9.4 mL protein solution with the mass concentration of 5-10 mg/mL, and adding the iridium complex solution into 55-55 mg/mL protein solution^oC, continuously stirring for 2-4 h, then adding 0.1 mL of alkali solution with the molar concentration of 0.1 mol/L to adjust the solution to a specific pH value, and adjusting the pH value to 37^oContinuously stirring for 12-24 hours under C to obtain a dark yellow precipitate, separating and purifying a product through ultrafiltration centrifugation, and dispersing the finally prepared iridium nanocrystals into 5-7 mL of phosphate buffer solution with the pH of 7.4 for storage;

(2) preparation of antibody-labeled iridium nanocrystal solution

Adding 50-70 mu L of cross-linking agent and 500-700 mu L of antibody standard solution (10 mu g/mL) into 1 mL of iridium nanocrystalline solution, incubating at 4 ℃ for 12-24 h, performing centrifugal separation to obtain an iridium nanocrystalline solution marked by an antibody, and storing at 4 ℃ for later use;

(3) preparation steps of iridium nanocrystalline electrochemiluminescence immunosensor

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 nanocrystal solution with the concentration of 2-3 mg/mL on the surface of the glassy carbon electrode, and airing the solution 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 type I procollagen amino-terminal propeptide solution of the multiple myeloma 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 the pH of 7.4, and finishing construction of the immunosensor;

(4) an iridium nanocrystalline electrochemiluminescence immunosensor is used for detecting a type I procollagen amino-terminal propeptide serving as a multiple myeloma marker.

2. The preparation method and application of the iridium nanocrystalline electrochemiluminescence sensor for detecting I-type procollagen amino-terminal propeptide according to claim 1, wherein the iridium complex is tris [2- (p-tolyl) pyridine-C2, N]Iridium Ir (mppy)₃。

3. The preparation and application of the iridium nano-crystal electrochemiluminescence sensor for detecting type I procollagen amino-terminal propeptide according to claim 1, wherein the protein solution is bovine serum albumin BSA solution.

4. The preparation and application of the iridium nanocrystalline electrochemiluminescence sensor for detecting type I procollagen amino-terminal propeptide according to claim 1, wherein the alkali solution is sodium hydroxide solution.

5. The preparation and application of the iridium nanocrystalline electrochemiluminescence sensor for detecting type I procollagen amino-terminal propeptide according to claim 1, wherein the specific pH value is 12.

6. The preparation and application of the iridium nanocrystalline electrochemiluminescence sensor for detecting type I procollagen amino-terminal propeptide according to claim 1, wherein the cross-linking agent is a glutaraldehyde solution with the mass fraction of 30%.

7. The iridium nanocrystal electrochemiluminescence immunosensor of claim 1 for detecting procollagen type i amino-terminal propeptide as a multiple myeloma marker, the electrochemoluminescence immunosensor comprising the following 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 polyethyleneimine to obtain corresponding electrochemiluminescence signal intensity when incubating I-type procollagen amino-terminal propeptides with different concentrations, and drawing a working curve (the detection limit is 35 fg/mL, and the linear range is 100 fg/mL-50 ng/mL);

(3) and testing a sensor for hatching an actual sample of the I-type procollagen amino-terminal propeptide with unknown concentration, and calculating according to the working curve to obtain the concentration of the I-type procollagen amino-terminal propeptide 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.

Multiple myeloma is a malignant tumor in which plasma cells proliferate clonally, causing bone damage, often accompanied by manifestation of bone disease. Therefore, it is important to find a noninvasive serum biological index for diagnosing and monitoring multiple myeloma. Researches find that the content of the type I procollagen amino-terminal pro-peptide PINP in serum of a patient in a progressive stage is higher than that of a patient in a remission stage, and the expression level of the type I procollagen amino-terminal pro-peptide PINP is obviously reduced along with the deepening of treatment, so that the method can be used for auxiliary diagnosis of multiple myeloma. However, immunoassay methods related thereto have been reported only rarely. Therefore, based on the superiority of electrochemical luminescence immunoassay, a simple, rapid and accurate electrochemical luminescence immunosensor is developed for measuring the I-type procollagen amino-terminal propeptide, and 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. The iridium complex is taken as the largest class in the complex family and has wide application in the development of organic electroluminescent devices. With extremely high electrochemiluminescence efficiency, with tris [2- (p-tolyl) pyridine-C2, N]Iridium Ir (mppy)₃Iridium as representativeComplexes have begun to attract attention in the field of bioassays. However, Ir (mppy)₃The water solubility is poor, the agglomeration is easy, the biocompatibility is poor, and the application of the water-soluble protein in water-phase immunoassay is greatly limited.

Disclosure of Invention

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

(2) the invention also aims to provide the application of the immunosensor, which is used for detecting the concentration of the type I procollagen amino-terminal propeptide of the multiple myeloma marker, has the advantages of rapid signal response and high sensitivity, greatly shortens the detection time, saves the manpower, material resources and financial resources, and has the detection limit of 35 fg/mL and the linear range of 100 fg/mL-50 ng/mL.

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

1. preparation and application of an iridium nanocrystalline electrochemical luminescence sensor for detecting I-type procollagen amino-terminal propeptide are characterized by comprising the following steps:

(1) preparation of Iridium nanocrystalline solution

Dissolving iridium complex solid with the mass of 10-20 mg in 1-4 mL tetrahydrofuran at room temperature to prepare iridium complex solution with the mass concentration of 5-10 mg/mL, measuring 0.5-1.5 mL iridium complex solution with the mass concentration of 5-10 mg/mL, dropwise adding the iridium complex solution with the mass concentration of 5-10 mg/mL into 8.4-9.4 mL protein solution with the mass concentration of 5-10 mg/mL, and adding the iridium complex solution into 55-55 mg/mL protein solution^oC, continuously stirring for 2-4 h, then adding 0.1 mL of alkali solution with the molar concentration of 0.1 mol/L to adjust the solution to a specific pH value, and adjusting the pH value to 37^oContinuously stirring for 12-24 hours under C to obtain a dark yellow precipitate, separating and purifying a product through ultrafiltration centrifugation, and dispersing the finally prepared iridium nanocrystals into 5-7 mL of phosphate buffer solution with the pH of 7.4 for storage;

the iridium complex is tris [2- (p-tolyl) pyridine-C2, N]Iridium Ir (mppy)₃；

The protein solution is bovine serum albumin BSA solution;

the alkali liquor is sodium hydroxide solution;

the value of the specific pH is 12.

(2) Preparation of antibody-labeled iridium nanocrystal solution

Adding 50-70 mu L of cross-linking agent and 500-700 mu L of antibody standard solution (10 mu g/mL) into 1 mL of iridium nanocrystalline solution, incubating at 4 ℃ for 12-24 h, performing centrifugal separation to obtain an iridium nanocrystalline solution marked by an antibody, and storing at 4 ℃ for later use;

the cross-linking agent is a glutaraldehyde solution with the mass fraction of 30%.

(3) Preparation steps of iridium nanocrystalline electrochemiluminescence immunosensor

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 nanocrystal solution with the concentration of 2-3 mg/mL on the surface of the glassy carbon electrode, and airing the solution 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 type I procollagen amino-terminal propeptide solution of the multiple myeloma 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 the pH of 7.4, and finishing construction of the immunosensor;

(4) an iridium nanocrystalline electrochemical luminescence immunosensor is used for detecting a type I procollagen amino-terminal propeptide of a multiple myeloma marker, and comprises 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 polyethyleneimine to obtain corresponding electrochemiluminescence signal intensity when incubating I-type procollagen amino-terminal propeptides with different concentrations, and drawing a working curve (the detection limit is 35 fg/mL, and the linear range is 100 fg/mL-50 ng/mL);

(3) and testing a sensor for hatching an actual sample of the I-type procollagen amino-terminal propeptide with unknown concentration, and calculating according to the working curve to obtain the concentration of the I-type procollagen amino-terminal propeptide in the actual sample.

Advantageous results of the invention

(1) The iridium nanocrystalline with good water solubility, good biocompatibility and high electrochemical luminescence efficiency is prepared for the first time, and the problems of poor water solubility and poor biocompatibility of the iridium complex in a water phase are solved;

(2) an iridium nanocrystalline solution marked by an antibody is used as a signal probe to develop a unmarked electrochemical luminescence immunosensor for detecting I-type procollagen amino-terminal propeptide of a multiple myeloma marker;

(3) the blank of the existing electrochemical luminescence detection method of the type I procollagen amino-terminal propeptide is made up, in the actual sample detection, the detection limit of the immunosensor is as low as 35 fg/mL, and the linear range is as wide as 100 fg/mL-50 ng/mL;

(4) the developed immunosensor has the advantages of simple preparation, easy operation, rapid signal response, good reproducibility, environmental protection, no pollution and the like, has strong specificity to the type I procollagen amino-terminal propeptide in serum, and has great potential application in early diagnosis of multiple myeloma.

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 iridium nanocrystalline electrochemical luminescence sensor for detecting I type procollagen amino-terminal propeptide, the iridium nanocrystalline solution has the following steps:

at room temperature, mixing the massIs 10 mg of tris [2- (p-tolyl) pyridine-C2, N]Iridium Ir (mppy)₃The solid was dissolved in 1 mL of tetrahydrofuran and prepared into Ir (mppy) at a mass concentration of 10 mg/mL₃The solution was measured and 0.5 mL of Ir (mppy) at a mass concentration of 10 mg/mL was added₃The solution was added dropwise to 8.4 mL of a bovine serum albumin BSA solution at a mass concentration of 5 mg/mL at 55^oC, after continuously stirring for 2 hours, adding 0.1 mL of alkali solution with the molar concentration of 0.1 mol/L to adjust the pH of the solution to 12, and adjusting the pH value to 37^oAnd C, continuously stirring for 12 hours to obtain a dark yellow precipitate, separating and purifying the product through ultrafiltration and centrifugation, and dispersing the finally prepared iridium nanocrystals into 5 mL of phosphate buffer solution with the pH of 7.4 for storage.

Embodiment 2. preparation and application of iridium nanocrystalline electrochemical luminescence sensor for detecting I type procollagen amino-terminal propeptide, the preparation steps of iridium nanocrystalline solution are as follows:

tris [2- (p-tolyl) pyridine-C2, N was added at room temperature in a mass of 15 mg]Iridium Ir (mppy)₃The solid was dissolved in 2 mL of tetrahydrofuran and prepared into Ir (mppy) at a mass concentration of 7.5 mg/mL₃The solution was measured out in 1 mL of Ir (mppy) at a mass concentration of 7.5 mg/mL₃The solution was added dropwise to 8.9 mL of 10 mg/mL bovine serum albumin BSA solution at 55^oC, after continuously stirring for 3 hours, adding 0.1 mL of alkali solution with the molar concentration of 0.1 mol/L to adjust the pH of the solution to 12, and adjusting the pH value to 37^oAnd C, continuously stirring for 18 h to obtain a dark yellow precipitate, separating and purifying the product through ultrafiltration and centrifugation, and dispersing the finally prepared iridium nanocrystals into 6 mL of phosphate buffer solution with the pH of 7.4 for storage.

Embodiment 3. preparation and application of iridium nanocrystalline electrochemical luminescence sensor for detecting I type procollagen amino-terminal propeptide, the preparation steps of iridium nanocrystalline solution are as follows:

tri [2- (p-tolyl) pyridine-C2, N was added at room temperature in a mass of 20 mg]Iridium Ir (mppy)₃The solid was dissolved in 4 mL of tetrahydrofuran and prepared into Ir (mppy) at a mass concentration of 5 mg/mL₃The solution was measured and 1.5 mL of Ir (mppy) at a mass concentration of 5 mg/mL was added₃The solution was added dropwise to 8.4 mL, 10 mg/mL bovine serum albumin BSA solution in 55^oC, after continuously stirring for 4 hours, adding 0.1 mL of alkali solution with the molar concentration of 0.1 mol/L to adjust the pH of the solution to 12, and adjusting the pH value to 37^oAnd C, continuously stirring for 24 hours to obtain a dark yellow precipitate, separating and purifying the product through ultrafiltration and centrifugation, and dispersing the finally prepared iridium nanocrystals into 7 mL of phosphate buffer solution with the pH of 7.4 for storage.

Embodiment 4. preparation and application of iridium nanocrystalline electrochemical luminescence sensor for detecting I type procollagen amino-terminal propeptide, iridium nanocrystalline solution marked by antibody is prepared according to the following steps:

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

Embodiment 5. preparation and application of iridium nanocrystalline electrochemical luminescence sensor for detecting I type procollagen amino-terminal propeptide, iridium nanocrystalline solution marked by antibody is prepared according to the following steps:

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

Embodiment 6. preparation and application of iridium nanocrystalline electrochemical luminescence sensor for detecting I type procollagen amino-terminal propeptide, iridium nanocrystalline solution marked by antibody is prepared according to the following steps:

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

Embodiment 7. preparation and application of an iridium nano-crystal electrochemical luminescence sensor for detecting I-type procollagen amino-terminal propeptide, the preparation 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 nanocrystalline 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. dripping 6 mu L of type I procollagen amino-terminal propeptide solution of the multiple myeloma 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 the pH of 7.4, and finishing construction of the immunosensor.

Embodiment 8. preparation and application of an iridium nano-crystal electrochemical luminescence sensor for detecting I-type procollagen amino-terminal propeptide, the preparation 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 nanocrystalline solution with the concentration of 3 mg/mL on the surface of the glassy carbon electrode, and placing the glassy carbon electrode at 37 ℃ for airing;

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. dripping 6 mu L of type I procollagen amino-terminal propeptide solution of the multiple myeloma 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 the pH of 7.4, and finishing construction of the immunosensor.

Embodiment 9. preparation and application of an iridium nano-crystal electrochemical luminescence sensor for detecting I-type procollagen amino-terminal propeptide, the preparation 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 nanocrystalline 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. dripping 6 mu L of type I procollagen amino-terminal propeptide solution of the multiple myeloma 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 the pH of 7.4, and finishing construction of the immunosensor.

Embodiment 10 an iridium nanocrystal electrochemiluminescence immunosensor for multiple myeloma marker type i procollagen amino-terminal propeptide, comprising the following 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 mmol/L polyethyleneimine to obtain corresponding electrochemiluminescence signal intensity when I-type procollagen amino-terminal propeptide with different concentrations is incubated, and drawing a working curve, wherein the detection limit is 35 fg/mL, and the linear range is 100 fg/mL-50 ng/mL;

(3) and testing a sensor for hatching an actual sample of the I-type procollagen amino-terminal propeptide with unknown concentration, and calculating according to the working curve to obtain the concentration of the I-type procollagen amino-terminal propeptide in the actual sample.

Embodiment 11, an iridium nanocrystal electrochemiluminescence immunosensor for multiple myeloma marker type i procollagen amino-terminal propeptide, comprising the following 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 35 mmol/L polyethyleneimine to obtain corresponding electrochemiluminescence signal intensity when I-type procollagen amino-terminal propeptide with different concentrations is incubated, and drawing a working curve, wherein the detection limit is 35 fg/mL, and the linear range is 100 fg/mL-50 ng/mL;

(3) and testing a sensor for hatching an actual sample of the I-type procollagen amino-terminal propeptide with unknown concentration, and calculating according to the working curve to obtain the concentration of the I-type procollagen amino-terminal propeptide in the actual sample.

Embodiment 12 an iridium nanocrystal electrochemiluminescence immunosensor for multiple myeloma marker type i procollagen amino-terminal propeptide, comprising the following 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 45 mmol/L polyethyleneimine to obtain corresponding electrochemiluminescence signal intensity when I-type procollagen amino-terminal propeptide with different concentrations is incubated, and drawing a working curve, wherein the detection limit is 35 fg/mL, and the linear range is 100 fg/mL-50 ng/mL;

(3) and testing a sensor for hatching an actual sample of the I-type procollagen amino-terminal propeptide with unknown concentration, and calculating according to the working curve to obtain the concentration of the I-type procollagen amino-terminal propeptide in the actual sample.