Novel electrochemiluminescence immunosensor for rapid and high sensitive detection of retinol binding protein and its preparation method

A technology for sensitive detection and binding of proteins, applied in the field of biosensors, can solve the problems of insufficient immunosensor signal, narrow linear range, low binding rate, etc., and achieves a simple and easy construction method, wide linear range and good reproducibility. Effect

Inactive Publication Date: 2013-08-07
SHANGHAI NORMAL UNIVERSITY
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Problems solved by technology

However, this method has certain limitations. Because the binding rate of ruthenium compounds to proteins or DNA is not high, the signals of most of these immunosensors are not strong enough, so the linear range of detection is narrow and the detection limit is low. , so it is difficult to apply to the detection of actual samples

Method used

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  • Novel electrochemiluminescence immunosensor for rapid and high sensitive detection of retinol binding protein and its preparation method
  • Novel electrochemiluminescence immunosensor for rapid and high sensitive detection of retinol binding protein and its preparation method
  • Novel electrochemiluminescence immunosensor for rapid and high sensitive detection of retinol binding protein and its preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Preparation of Double Antibody Sandwich Type Immunosensor

[0025] 1) 10mg of synthesized SiO 2 Nanoparticles were dispersed in 5mL Nafion (5wt%)-ethanol solution and stirred in an ice bath for 4 hours to obtain Nafion-coated SiO 2 (NafionSiO 2 ) solution, the solution is centrifuged (rotating speed is 10000rpm, temperature is 4°C);

[0026] 2) Disperse the obtained solid particles in 2mL PBS (0.01M) (pH7.4), inject 600mL mouse anti-RBP (39μg / mL), stir continuously in an ice bath for 6 hours, centrifuge and dissolve in 1mL pH=7.4 PBS (0.01M);

[0027] 3) Add 1mL of 30mM Ru(bpy) 3 Cl 2 Add to 1 mL of the above NafionSiO with secondary antibody 2 Suspension of Ru(bpy) in the ice bath was continuously stirred for 1 hour, and the Ru(bpy) 3 Cl 2 Adsorbed to NafionSiO linked to secondary antibody 2 The surface of the complex was centrifuged to obtain RBP secondary antibody-labeled Ru–NafionSiO 2 Composite nanoparticles;

[0028] 4) The obtained composite...

Embodiment 2

[0035] Example 2: Ru–NafionSiO labeled with RBP monoclonal antibody 2 Electrochemical Behavior of Glassy Carbon Electrodes Modified by Composite Nanoparticles and Multi-walled Carbon Nanotubes

[0036] 10 μL RBP secondary antibody and 10 μL RBP secondary antibody-labeled Ru–NafionSiO were respectively modified on the glassy carbon electrode modified with multi-walled carbon nanotubes 2 The composite nanoparticle solution was tested for electrochemical behavior in 4mL PBS (pH8.0) detection cell containing 0.25mM TPA. There was basically no electrical signal between the single RBP antibody and the electrode modified by multi-walled carbon nanotubes, but the RBP secondary antibody labeled Ru–NafionSiO 2 The peak current of the electrode modified by composite nanoparticles and multi-walled carbon nanotubes is significantly increased, and there is a pair of Ru(bpy) 3 2+ The characteristic redox peak of ( figure 2 ), indicating that RBP secondary antibody-labeled Ru–NafionSiO ...

Embodiment 3

[0037] Example 3: Electrochemical Impedance Characterization of the Construction Process of Double Antibody Sandwich Type Immunosensor

[0038] During the modification process of the electrode, it was characterized by electrochemical impedance method. First place the treated bare glassy carbon electrode in 4mL K 3 Fe(CN) 6 / K 4 Fe(CN) 6 In impedance solution (5mM), the impedance value is very small, as the electrode surface is modified layer by layer with RBP primary antibody, BSAT, antigen, and RBP secondary antibody-labeled Ru–NafionSiO 2 The impedance value of the composite nanoparticles also increases, indicating that the modifiers are well fixed on the electrode surface.

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Abstract

The invention discloses a novel electrochemiluminescence immunosensor for rapid and high sensitive detection of retinol binding protein. By adopting a double-antibody sandwich method, and by making use of the large specific surface area and good electrical conductivity of a multiwalled carbon nanotube and good biocompatibility of SiO2, a regular surface porous structure, high adsorption capacity and other advantages, the dual signal amplified electrochemiluminescence immunosensor can be constructed on a glassy carbon electrode surface and can perform electrochemiluminescence detection on RBP (retinol binding protein) in the human body fluid. Results show that, by employing an electrochemiluminescence immunosensing method, the RBP detection concentration range is wide, the detection limit is low, and the immunosensor has good stability, reproducibility and other sensing properties.

Description

technical field [0001] The invention relates to biosensor technology, in particular to a novel electrochemiluminescent immunosensor for rapid and highly sensitive detection of retinol-binding protein and a preparation method thereof. Background technique [0002] Clinically, early diagnosis of disease plays a vital role in improving the possibility of disease cure, which brings certain challenges to the analysis methods of various disease markers, and also promotes the rapid development of sensor research for detection of disease markers. Electrochemiluminescence refers to an electrochemical reaction in which a substance undergoes a high-energy electron transfer reaction on an electrode to form an excited state to emit light. It combines the dual advantages of electrochemistry and optics. In recent years, it has gradually developed into a A new analytical test method applied in many fields such as biomedical detection. Electrochemiluminescence immunosensor has also become a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/53
Inventor 贾能勤吴贝娜胡宸溢
Owner SHANGHAI NORMAL UNIVERSITY
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