CN112683968B - Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot - Google Patents
Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot Download PDFInfo
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Abstract
The invention relates to a preparation method of an electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots, which is characterized in that iridium nanodots with good water solubility, good biocompatibility and high electrochemical luminescence efficiency are prepared, and an antibody-marked iridium nanodot solution is used as a signal probe to develop a label-free electrochemical luminescence immunosensor which is used for rapid and sensitive detection of a interstitial lung disease marker sialylated sugar chain antigen KL-6, wherein the detection limit is 45 fg/mL, the linear range is 100 fg/mL-50 ng/mL, and the preparation method has the advantages of strong specificity, good reproducibility, stable signal and the like.
Description
Technical Field
The invention belongs to the technical fields of nano materials and biological sensing.
Background
As a research hot spot which is created 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 the immunoassay method, and the prepared immunosensor has the advantages of low cost, good selectivity, high sensitivity, high analysis speed, easiness in automation, micromation and integration and the like, and has been 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 concealment, long disease course, complex disease mechanism, great difficulty in diagnosis and treatment and the like. Such diseases cause damage to lung organs and corresponding dysfunction, and have become a hot spot and difficult problem of clinical concern in recent years. Bronchoscopy and surgical lung biopsy are not suitable for repeated examination in a short period, and the lung function examination needs a high degree of patient coordination, has a plurality of influence factors and is not suitable for routine detection of severe patients. It follows that it is important to find a serum noninvasive biological index 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 rarely reported. Therefore, based on the superiority of electrochemiluminescence immunoassay, a simple, rapid and accurate electrochemiluminescence immunosensor is developed for determining sialylated sugar chain antigen KL-6, and has important research significance and clinical application value.
In recent years, the design and synthesis of phosphorescent transition metal complexes has become a research hotspot. Iridium complexes are the largest class of complex families, and have wide application in the development of organic electroluminescent devices. Ir (ppy) iridium acetylacetonate with bis (2-phenylpyridine) by virtue of extremely high electrochemiluminescence efficiency 2 Iridium complexes represented by (acac) have been attracting attention in the field of bioassays. Ir (ppy) 2 (acac) has poor water solubility, easy agglomeration and poor biocompatibility, and greatly limits the application of the (acac) in aqueous phase immunoassay.
Disclosure of Invention
(1) One of the purposes of the invention is to overcome the defects, prepare an iridium nano dot with good water solubility, good biocompatibility and high electrochemical luminescence efficiency, and develop a label-free electrochemical luminescence immunosensor by taking an iridium nano dot solution marked by an antibody as a signal probe;
(2) The second purpose of the invention is to provide the application of the immunosensor, which is used for detecting the concentration of the sialylated sugar chain antigen KL-6 of the marker of the interstitial lung disease, the sensor has rapid signal response and high sensitivity, greatly shortens the detection time, saves manpower, material resources and financial resources, and has the detection limit of 45 fg/mL and the linear range of 100 fg/mL-50 ng/mL.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a preparation method of an electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots comprises the following steps:
1. preparation of iridium nanodot solution
At room temperature, dissolving iridium complex solid with mass of 10-20 mg in 1-4 mL tetrahydrofuran to prepare a solution with mass concentration of 5-10 mg/mL; uniformly oscillating a sodium hydroxide solution with the molar concentration of 0.1-0.3 mol/L and a deferiprone 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 deferiprone structural units into free subunits; then, dropwise adding 0.5-1.5 mL iridium complex solution with mass concentration of 5-10 mg/mL, continuously stirring for 2-4 h, and rapidly adding 0.1-0.3 mL hydrochloric acid solution with molar concentration of 0.1-0.3 mol/L into the mixed solution to neutralize the solution; after the mixed solution is kept still for 2-4 h, separating and purifying the product by an ultrafiltration centrifuge tube, and dispersing the finally prepared iridium nanodots into a phosphate buffer solution with the pH value of 7.4 and 5-7 mL for later use;
the iridium complex is bis (2-phenylpyridine) iridium acetylacetonate Ir (ppy) 2 (acac)；
The deferiprone is biotin marked horse spleen deferiprone;
the volume ratio of the sodium hydroxide solution to the deferiprone solution is 1:100.
2. Preparation of antibody-labeled iridium nanodot solution
50-70 mu L of glutaraldehyde with the mass fraction of 30% and 10 mu g/mL of antibody standard solution with the concentration of 10 mu g/mL are added into the 1 mL iridium nanodot solution, after incubation is carried out at 4 ℃, 12-24 h of antibody-labeled iridium nanodot solution is obtained through centrifugal separation, and the solution is placed at 4 ℃ for storage for standby;
3. preparation of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot
a. Polishing the glassy carbon electrode with the diameter of 4 mm by using alumina polishing powder with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and flushing the glassy carbon electrode 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 a glassy carbon electrode, and airing at 37 ℃;
c. dripping 3 mu L of bovine serum albumin solution with the mass fraction of 1-3% to seal nonspecific active sites on the surface of the electrode, flushing the surface of the electrode by phosphate buffer solution PBS with the pH of 7.4, and airing the electrode at the temperature of 4 ℃;
d. dripping 6 mu L of a sialylated sugar chain antigen KL-6 solution of an interstitial lung disease marker with the concentration of 10 mu g/mL or unknown concentration, placing the solution in 37 ℃ for incubation of 1 h, flushing the surface of an electrode by using phosphate buffer solution PBS with the pH of 7.4, and finishing the construction of an immunosensor;
4. the iridium nano-dot electrochemiluminescence immunosensor is used for detecting a marker sialylated sugar chain antigen KL-6 of interstitial lung diseases, and comprises the following operation steps:
(1) Parameter setting: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to 800V, the cyclic voltammetry scanning potential range of an electrochemical workstation is set to 0-1.0V, and the scanning rate is set to 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, performing electrochemiluminescence test in a phosphate buffer solution containing 25-45 mmol/L triethylamine in 10 mL to obtain electrochemiluminescence signal intensities corresponding to incubation of sialylated sugar chain antigens KL-6 with different concentrations, 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 a sensor for incubating an actual sample with sialylated sugar chains of unknown concentration, and calculating according to the working curve to obtain the sialylated sugar chain concentration in the actual sample.
Advantageous results of the invention
(1) The method for efficiently encapsulating iridium complex molecules in the deferiprone is provided for the first time, and the problems of poor water solubility and poor biocompatibility of the iridium complex in water are solved when the iridium nano-meter with good water solubility and high electrochemiluminescence efficiency is prepared;
(2) An iridium nano dot solution marked by an antibody is used as a signal probe to develop a label-free electrochemiluminescence immunosensor which is used for detecting a sialylated sugar chain antigen KL-6 of a marker of interstitial lung diseases;
(3) The blank of the existing sialylated sugar chain antigen KL-6 electrochemiluminescence detection method is made up, 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 in actual sample detection;
(4) The developed immunosensor has the advantages of simple preparation, easy operation, rapid signal response, good reproducibility, green environmental protection, no pollution and the like, has strong specificity for sialylated sugar chain antigen KL-6 in serum, and has huge potential application in early diagnosis of interstitial lung diseases.
Detailed Description
The invention will be further illustrated with reference to specific examples, which are to be understood as illustrative only and are not intended to limit the scope of the invention.
Embodiment 1. A method for preparing an electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots, the steps of the iridium nanodot solution are as follows:
ir (ppy) iridium bis (2-phenylpyridine) acetylacetonate having a mass of 10 mg was introduced at room temperature 2 (acac) solids were dissolved in 1 mL tetrahydrofuran to make 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 deferiprone solution with the mass concentration of 0.5 mg/mL according to the volume ratio of 1:100, and continuously stirring for 2 h at room temperature to completely dissociate deferiprone structural units into free subunits; then, 0.5 mL iridium complex solution with the mass concentration of 10 mg/mL is added dropwise, after continuous stirring is carried out for 2 h, 0.1 mL hydrochloric acid solution with the molar concentration of 0.1 mol/L is rapidly added into the mixed solution to neutralize the solution; after the mixed solution is stopped for 2 h, an ultrafiltration centrifuge tube is usedAnd (3) separating and purifying the product, and dispersing the finally prepared iridium nano dots into a phosphate buffer solution with the pH of 7.4 and 5 mL for storage.
Example 2. Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot, the preparation steps of iridium nanodot solution are as follows:
ir (ppy) iridium bis (2-phenylpyridine) acetylacetonate having a mass of 15 mg was introduced at room temperature 2 (acac) dissolved in 2 mL 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 deferiprone solution with the mass concentration of 0.7: 0.7 mg/mL according to the volume ratio of 1:100, and continuously stirring for 3 h at room temperature to completely dissociate deferiprone structural units into free subunits; then, 1. 1 mL iridium complex solution with the mass concentration of 7.5 mg/mL is added dropwise, after continuous stirring for 3 h, 0.2 mL hydrochloric acid solution with the molar concentration of 0.2 mol/L is rapidly added into the mixed solution to neutralize the solution; after the mixed solution is static for 3 h, separating and purifying the product by an ultrafiltration centrifuge tube, and dispersing the finally prepared iridium nanodots into a phosphate buffer solution with the pH value of 7.4 and 6 mL for storage for later use.
Embodiment 3. Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot, the preparation steps of iridium nanodot solution are as follows:
ir (ppy) iridium acetylacetonate (bis (2-phenylpyridine) having a mass of 20 mg was introduced at room temperature 2 (acac) dissolved in 4 mL 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 deferiprone solution with the mass concentration of 5 mg/mL according to the volume ratio of 1:100, and continuously stirring for 4 h at room temperature to completely dissociate deferiprone structural units into free subunits; then, 1.5 mL iridium complex solution with the mass concentration of 5 mg/mL is dropwise added, after continuously stirring for 4 h, 0.3 mL hydrochloric acid solution with the molar concentration of 0.3 mol/L is rapidly added into the mixed solution to neutralize the solution; after the mixed solution is kept at a standstill of 4 h, separating and purifying the product by an ultrafiltration centrifuge tube, and finally obtaining the iridium nanodotsDispersing into phosphate buffer solution with pH of 7.4 and 7. 7 mL, and storing for standby.
Example 4. Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot, the iridium nanodot solution marked by antibody is prepared according to the following steps:
taking 1 mL of the iridium nanodot solution prepared above, 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 the iridium nanodot solution marked by the antibody through centrifugal separation, and storing at 4 ℃ for later use.
Example 5. Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot, antibody-labeled iridium nanodot solution was prepared according to the following steps:
taking 1 mL of the iridium nanodot solution prepared above, 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 at 4 ℃ for 18 h, obtaining the iridium nanodot solution marked by the antibody through centrifugal separation, and storing at 4 ℃ for later use.
Example 6. Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot, the iridium nanodot solution marked by antibody is prepared according to the following steps:
taking 1 mL of the iridium nanodot solution prepared above, 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 the iridium nanodot solution marked by the antibody through centrifugal separation, and storing at 4 ℃ for later use.
Example 7. Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot, the construction steps of the immunosensor are as follows:
a. polishing the glassy carbon electrode with the diameter of 4 mm by using alumina polishing powder with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and flushing the glassy carbon electrode 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 a glassy carbon electrode, and airing at 37 ℃;
c. dripping 3 mu L of bovine serum albumin solution with the mass fraction of 1% to seal nonspecific active sites on the surface of the electrode, flushing the surface of the electrode with phosphate buffer solution PBS with the pH of 7.4, and airing at 4 ℃;
d. and (3) dripping 6 mu L of a saliva acidized 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, flushing the surface of the electrode by a phosphate buffer solution PBS with the pH of 7.4, and completing the construction of the immunosensor.
Example 8. A method for preparing 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 the glassy carbon electrode with the diameter of 4 mm by using alumina polishing powder with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and flushing the glassy carbon electrode 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 a glassy carbon electrode, and airing at 37 ℃;
c. dripping 3 mu L of bovine serum albumin solution with the mass fraction of 2% to seal nonspecific active sites on the surface of the electrode, flushing the surface of the electrode with phosphate buffer solution PBS with the pH of 7.4, and airing at 4 ℃;
d. and (3) dripping 6 mu L of a saliva acidized 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, flushing the surface of the electrode by a phosphate buffer solution PBS with the pH of 7.4, and completing the construction of the immunosensor.
Example 9. Preparation method of electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot, the construction steps of the immunosensor are as follows:
a. polishing the glassy carbon electrode with the diameter of 4 mm by using alumina polishing powder with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and flushing the glassy carbon electrode 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 a glassy carbon electrode, and airing at 37 ℃;
c. dripping 3 mu L of bovine serum albumin solution with the mass fraction of 3% to seal nonspecific active sites on the surface of the electrode, flushing the surface of the electrode with phosphate buffer solution PBS with the pH of 7.4, and airing at 4 ℃;
d. and (3) dripping 6 mu L of a saliva acidized 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, flushing the surface of the electrode by a phosphate buffer solution PBS with the pH of 7.4, and completing the construction of the immunosensor.
Example 10. An iridium nanodot electrochemiluminescence immunosensor is used for sialylated sugar chain antigen KL-6, which is a marker of interstitial lung diseases, and comprises the following operation steps:
(1) Parameter setting: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to 800V, the cyclic voltammetry scanning potential range of an electrochemical workstation is set to 0-1.0V, and the scanning rate is set to 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, performing electrochemiluminescence test in a phosphate buffer solution containing 25 mmol/L triethylamine in 10 mL to obtain electrochemiluminescence signal intensities corresponding to incubation of sialylated saccharide chain antigens KL-6 with different concentrations, and 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 a sensor for incubating an actual sample with sialylated sugar chains of unknown concentration, and calculating according to the working curve to obtain the sialylated sugar chain concentration in the actual sample.
Example 11. An iridium nanodot electrochemiluminescence immunosensor is used for sialylated sugar chain antigen KL-6, which is a marker of interstitial lung diseases, and comprises the following operation steps:
(1) Parameter setting: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to 800V, the cyclic voltammetry scanning potential range of an electrochemical workstation is set to 0-1.0V, and the scanning rate is set to 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, performing electrochemiluminescence test in a phosphate buffer solution containing 35 mmol/L triethylamine in 10 mL to obtain electrochemiluminescence signal intensities corresponding to incubation of sialylated saccharide chain antigens KL-6 with different concentrations, and 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 a sensor for incubating an actual sample with sialylated sugar chains of unknown concentration, and calculating according to the working curve to obtain the sialylated sugar chain concentration in the actual sample.
Example 12. An iridium nanodot electrochemiluminescence immunosensor is used for sialylated sugar chain antigen KL-6, which is a marker of interstitial lung diseases, and comprises the following operation steps:
(1) Parameter setting: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to 800V, the cyclic voltammetry scanning potential range of an electrochemical workstation is set to 0-1.0V, and the scanning rate is set to 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, performing electrochemiluminescence test in a phosphate buffer solution containing 45 mmol/L triethylamine in 10 mL to obtain electrochemiluminescence signal intensities corresponding to incubation of sialylated saccharide chain antigens KL-6 with different concentrations, and 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 a sensor for incubating an actual sample with sialylated sugar chains of unknown concentration, and calculating according to the working curve to obtain the sialylated sugar chain concentration in the actual sample.
Claims (4)
1. The preparation method of the electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots is characterized by comprising the following steps of:
(1) Preparation of iridium nanodot solution
At room temperature, dissolving iridium complex solid with mass of 10-20 mg in 1-4 mL tetrahydrofuran to prepare a solution with mass concentration of 5-10 mg/mL; uniformly oscillating a sodium hydroxide solution with the molar concentration of 0.1-0.3 mol/L and a deferiprone 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 deferiprone structural units into free subunits; then, dropwise adding 0.5-1.5 mL iridium complex solution with mass concentration of 5-10 mg/mL, continuously stirring for 2-4 h, and rapidly adding 0.1-0.3 mL hydrochloric acid solution with molar concentration of 0.1-0.3 mol/L into the mixed solution to neutralize the solution; after the mixed solution is kept still for 2-4 h, separating and purifying the product by an ultrafiltration centrifuge tube, and dispersing the finally prepared iridium nanodots into a phosphate buffer solution with the pH value of 7.4 and 5-7 mL for later use;
(2) Preparation of antibody-labeled iridium nanodot solution
50-70 mu L of glutaraldehyde with the mass fraction of 30% and 500-700 mu L of antibody standard solution with the concentration of 10 mu g/mL are added into the 1 mL iridium nanodot solution, incubated at 4 ℃ for 12-24 h, the iridium nanodot solution marked by the antibody is obtained through centrifugal separation, and the iridium nanodot solution is placed at 4 ℃ for storage for standby;
(3) Preparation step of iridium nano-dot electrochemical luminescence sensor
a. Polishing the glassy carbon electrode with the diameter of 4 mm by using alumina polishing powder with the diameters of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, and flushing the glassy carbon electrode 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 a glassy carbon electrode, and airing at 37 ℃;
c. dripping 3 mu L of bovine serum albumin solution with the mass fraction of 1-3% to seal nonspecific active sites on the surface of the electrode, flushing the surface of the electrode by phosphate buffer solution PBS with the pH of 7.4, and airing the electrode at the temperature of 4 ℃;
d. dripping 6 mu L of a sialylated sugar chain antigen KL-6 solution of an interstitial lung disease marker with the concentration of 10 mu g/mL or unknown concentration, placing the solution in 37 ℃ for incubation of 1 h, flushing the surface of an electrode by using phosphate buffer solution PBS with the pH of 7.4, and finishing the preparation of the immunosensor;
(4) The electrochemiluminescence immunosensor prepared in the step (3) is used for detecting a sialylated sugar chain antigen KL-6 serving as a marker of interstitial lung diseases, and the detection steps are as follows:
a. parameter setting: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to 800V, the cyclic voltammetry scanning potential range of an electrochemical workstation is set to 0-1.0V, and the scanning rate is set to 0.1V/s;
b. and (3) testing: taking a silver/silver chloride electrode as a reference electrode and a platinum wire electrode as a counter electrode, performing electrochemiluminescence test in a phosphate buffer solution containing 25-45 mmol/L triethylamine in 10 mL to obtain electrochemiluminescence signal intensities corresponding to incubating different concentrations of sialylated sugar chain antigen KL-6, drawing a working curve, wherein the detection limit is 45 fg/mL, and the linear range is 100 fg/mL-50 ng/mL;
c. and testing a sensor for incubating an actual sample with sialylated sugar chains of unknown concentration, and calculating according to the working curve to obtain the sialylated sugar chain concentration in the actual sample.
2. The method for preparing the electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodot according to claim 1, characterized in that the iridium complex is bis (2-phenylpyridine) iridium acetylacetonate Ir (ppy) 2 (acac)。
3. The method for preparing the electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots according to claim 1, characterized in that the deferiprone is biotin-labeled horse spleen deferiprone.
4. The method for preparing the electrochemical luminescence sensor for detecting sialylated sugar chain antigen KL-6 iridium nanodots according to claim 1, characterized in that the solution volume ratio of sodium hydroxide to deferiprone is 1:100.
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