Preparation method and application of apoferritin-based encapsulated Ru (bpy) 3<2+> immunosensor

A technology of apoferritin and immunosensor, which is applied in the field of new nanomaterials and biosensing, can solve the problems of signal stability, poor reproducibility, poor biocompatibility, adverse effects on accuracy, etc., and achieve high sensitivity , reproducible, easy to prepare effects

Inactive Publication Date: 2020-10-13
UNIV OF JINAN
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0004] due to Ru(bpy) 3 2+ It is difficult to fix on the surface of the glassy carbon electrode, resulting in poor signal stability and reproducibility. The traditional system usually introduces other new nanomaterials to solve this problem, but this will make the sensor construction process complicated and cumbersome. Poor compatibility, which is not conducive to the incubation of antigens and antibodies, and thus adversely affects the accuracy of the sensor's detection results, making it difficult to achieve clinical application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1. A kind of encapsulation Ru(bpy) based on apoferritin 3 2+ The preparation method and application of the immunosensor is characterized in that it comprises the following steps:

[0025] (1) Polish the glassy carbon electrode with a diameter of 4 mm with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powder in sequence, and rinse it with ultrapure water;

[0026] (2) Drop-coat 6 μL of CYFRA 21-1 antibody-labeled Ru(bpy) at a concentration of 5 mg / mL on the surface of the glassy carbon electrode 3 2+ The @apoferritin solution was used as the sensing substrate, which was dried at 37 °C;

[0027] (3) Add 3 μL bovine serum albumin solution with a mass fraction of 1% dropwise to block the non-specific active sites on the electrode surface, rinse the electrode surface with pH 7.4 phosphate buffer solution PBS, and place it at 4 °C C to dry;

[0028] (4) Add 6 μL of CYFRA 21-1 antigen solution with a concentration of 10 μg / mL or an unknown concentration, incuba...

Embodiment 2

[0029] Example 2.1. A kind of encapsulation Ru(bpy) based on apoferritin 3 2+ The preparation method and application of the immunosensor is characterized in that it comprises the following steps:

[0030] (1) Polish the glassy carbon electrode with a diameter of 4 mm with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powder in sequence, and rinse it with ultrapure water;

[0031](2) Drop-coat 6 μL of CYFRA 21-1 antibody-labeled Ru(bpy) at a concentration of 10 mg / mL on the surface of the glassy carbon electrode 3 2+ The @apoferritin solution was used as the sensing substrate, which was dried at 37 °C;

[0032] (3) Add 3 μL of bovine serum albumin solution with a mass fraction of 2% dropwise to block the non-specific active sites on the electrode surface, rinse the electrode surface with pH 7.4 phosphate buffer solution PBS, and place it at 4 °C C to dry;

[0033] (4) Add 6 μL of CYFRA 21-1 antigen solution with a concentration of 10 μg / mL or an unknown concentration, incu...

Embodiment 3

[0034] Example 3.1. A kind of encapsulation Ru(bpy) based on apoferritin 3 2+ The preparation method and application of the immunosensor is characterized in that it comprises the following steps:

[0035] (1) Polish the glassy carbon electrode with a diameter of 4 mm with 1.0 μm, 0.3 μm, and 0.05 μm alumina polishing powder in sequence, and rinse it with ultrapure water;

[0036] (2) Drop-coat 6 μL of CYFRA 21-1 antibody-labeled Ru(bpy) at a concentration of 15 mg / mL on the surface of the glassy carbon electrode 3 2+ The @apoferritin solution was used as the sensing substrate, which was dried at 37 °C;

[0037] (3) Add 3 μL bovine serum albumin solution with a mass fraction of 3% dropwise to block the non-specific active sites on the electrode surface, rinse the electrode surface with pH 7.4 phosphate buffer solution PBS, and place it at 4 °C C to dry;

[0038] (4) Add 6 μL of CYFRA 21-1 antigen solution with a concentration of 10 μg / mL or an unknown concentration, incuba...

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Abstract

The invention discloses a preparation method and application of an apoferritin-based encapsulated Ru (bpy) 3<2+> immunosensor, and belongs to the field of nano materials and the field of biosensing. On the basis of the structural characteristics of uniform morphology, hydrothermal resistance and controllable assembly of apoferritin and the excellent electrochemiluminescence property of Ru (bpy) 3<2+>, a thermal induction method is provided for the first time to realize efficient and rapid packaging of a large amount of Ru (bpy) 3<2+> molecules in apoferritin and thus the compound Ru (bpy) 3<2+> apoferritin with excellent biocompatibility and high luminous efficiency is successfully prepared. The method is simple and efficient, the harsh pH value requirement and complex material synthesis steps of a traditional packaging method can be avoided, and the innovativeness is obvious. Antibody molecules are modified on the surface of Ru (bpy) 3<2+> @apoferritin; a label-free electrochemiluminescence immunosensor for detection of non-small cell lung cancer disease marker CYFRA 21-1 is researched and developed; the detection limit is as low as 31 pg / mL, the linear range is 100 pg / mL-50 ng / mL, and the immunosensor has an obvious potential application value in similar detection methods.

Description

technical field [0001] The invention belongs to the field of novel nanometer materials and the technical field of biosensing. Background technique [0002] CYFRA 21-1 is a soluble fragment of cytokeratin 19, which is considered to be a tumor marker mainly used in the detection of lung cancer, especially for the diagnosis of non-small cell lung cancer. If there are unclear circular shadows in the lungs and the serum CYFRA21-1 concentration is higher than 30 ng / mL, the possibility of primary bronchial lung cancer is very high, and the positive detection rate of various types of non-small cell lung cancer is 70% to 85%. The serum concentration of CYFRA21-1 is positively correlated with the clinical stage of the tumor, and it can also be used as an effective indicator for tracking early recurrence of lung cancer after surgery, radiotherapy and chemotherapy. A high serum concentration of CYFRA 21-1 indicates that the disease is in an advanced stage and the prognosis is poor. The...

Claims

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Application Information

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IPC IPC(8): G01N27/48G01N21/76G01N27/30G01N27/327G01N33/68G01N33/574
CPCG01N27/48G01N21/76G01N27/3277G01N27/308G01N33/6893G01N33/57423G01N33/57484
Inventor 杨磊魏琴孙强杜宇吴丹王欢任祥马洪敏
Owner UNIV OF JINAN
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