Preparation method and application of avian leukemia virus antigen immunosensor

A technology for avian leukosis virus and antigen immunization, which is applied in the field of immune analysis and biosensing, can solve the problems of time-consuming, etc., and achieve the effects of less reagent consumption, simple preparation, and easy miniaturization

Inactive Publication Date: 2015-03-25
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are often time-consuming, in contrast, sandwich-type electrochemical immunoassay has become an important...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Core-shell nanomaterial amino-β-CDFe 3 o 4 SiO 2 - Preparation of Fc

[0028] 2.5g of Fe 3 o 4 ·7H 2 O with 4.0g Fe 2 (SO 4 ) 3Dissolve in a 500mL flask filled with 200mL of ultrapure water, add 50mL of ammonia water with a mass fraction of 25% to adjust the pH to 10, stir at a constant temperature of 45 °C for 1 h, and cool to room temperature; add 8 mL of tetraethyl ortho silicon salt TEOS, stirred for 16 h, centrifuged, and vacuum-dried at 50°C for 12 h to obtain Fe 3 o 4 SiO 2 ;

[0029] Take 0.08g Fe 3 o 4 SiO 2 Mix with 0.1 mL of aminopropyltriethoxysilane APTES and 10 mL of toluene, reflux at 110 °C for 1.5 h, and magnetically separate with a strong magnet. The obtained product is washed with methanol and ethanol three times in turn, and dried in vacuum for 12 h to obtain Aminated Fe 3 o 4 SiO 2 ;

[0030] 40 mg of aminated Fe 3 o 4 SiO 2 Disperse into 1 mL of absolute ethanol, add 250-350 μL of glutaraldehyde with a mass fraction ...

Embodiment 2

[0031] Example 2 Core-shell nanomaterial amino-β-CDFe 3 o 4 SiO 2 - Preparation of Fc

[0032] 3.5g of Fe 3 o 4 ·7H 2 O with 4.0g Fe 2 (SO 4 ) 3 Dissolve in a 500mL flask filled with 200mL of ultrapure water, add 50mL of ammonia water with a mass fraction of 28% to adjust the pH to 10, stir at a constant temperature of 55°C for 1 h, and cool to room temperature; add 12 mL of tetraethyl ortho silicon salt TEOS, stirred for 20 h, centrifuged, and vacuum-dried at 50°C for 12 h to obtain Fe 3 o 4 SiO 2 ;

[0033] Take 0.12g Fe 3 o 4 SiO 2 Mix with 0.1 mL of aminopropyltriethoxysilane APTES and 10 mL of toluene, reflux at 110 °C for 1.5 h, and perform magnetic separation with a strong magnet. The obtained product is washed with methanol and ethanol four times in turn, and dried in vacuum for 12 h to obtain Aminated Fe 3 o 4 SiO 2 ;

[0034] 60 mg of aminated Fe 3 o 4 SiO 2 Disperse into 1 mL of absolute ethanol, add 350 μL of glutaraldehyde with a mass fractio...

Embodiment 3

[0035] Example 3 Amination β -CDFe 3 o 4 SiO 2 -Fc / Ab 2 preparation

[0036] Aminated 3.5 mg β -CDFe 3 o 4 SiO 2 -Fc was dispersed in 1 mL of PBS solution with pH 7.0, sonicated, and aminated β -CDFe 3 o 4 SiO 2 -Fc suspension, add 10 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride EDC and 10 mg of N-hydroxysuccinimide NHS, shake at room temperature for 15 min, Add 0.5 mL, 80 μg·mL -1 detection antibody Ab 2 The solution was incubated at 4°C with shaking for 12 h, centrifuged to discard the supernatant, washed and separated with PBS solution with pH=7.0, added 200 μL of PBS solution, and placed in a refrigerator at 4°C for use.

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Abstract

The invention belongs to the technical field of immunoassay and bio-sensing, and discloses a preparation method and an application of an avian leukemia virus antigen immunosensor which is used for quickly detecting the avian leukemia virus antigen. According to the manufacturing scheme adopted by the invention, the preparation method comprises the following steps: by taking a glassy carbon electrode as a working electrode, modifying beta-CD/MWCNTs; then, adding adamantanecarboxylic acid/capturing antibody Ab1, bovine serum albumin, avian leukemia virus antigen as well as beta-CD@Fe3O4@SiO2-Fc/Ab2 solution in sequence. The beta-CD@Fe3O4@SiO2-Fc is easy to separate due to the superparamagnetism of a ferric oxide nano material; by utilizing the supramolecular recognition characteristic of the cyclodextrin, a great deal of ferrocenecarboxylic acid Fc can be captured through the action of subjective and objective objects, the biocompatibility and the water solubility are good, so that the preparation method is beneficial for increasing the antibody detecting amount, capable of realizing relatively high sensitivity and lowering the detecting limit to 0.33pg/mL.

Description

technical field [0001] The invention belongs to the technical field of immune analysis and biosensing, in particular to a β-CDFe-based 3 o 4 SiO 2 - Preparation and application of electrochemical immunosensor for Fc, a sandwich type electrochemical immunosensor for rapid detection of avian leukosis virus antigen was developed. Background technique [0002] Avian leukosis virus is a retrovirus group characterized by mainly causing neoplastic proliferation of various hematopoietic cells in poultry. Among them, subgroup J avian leukosis virus ALV-J is a retrovirus that mainly infects broiler chickens. Since the 1990s, it has had a huge impact on poultry animals worldwide, causing huge economic losses. A series of analytical methods have been devoted to the detection of ALV-J, such as immunohistochemistry, enzyme-linked immunosorbent assay, and surface plasmon resonance. However, these methods are often time-consuming. In contrast, sandwich-type electrochemical immunoassay h...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/531G01N27/327
CPCG01N27/3278G01N33/54366G01N33/54386G01N33/56983G01N33/57426
Inventor 颜兆庆魏琴高健吴丹闫涛张铁范大伟胡丽华
Owner UNIV OF JINAN
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