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A Protein Immobilization Method Based on Surface Plasmon Resonance Imaging Biosensor Chip

A surface plasmon and protein immobilization technology, applied in the field of protein chip arrays, can solve the problems of ineffectiveness and antibody inactivation, achieve high detection signal and avoid inactivation

Active Publication Date: 2017-09-05
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In such cases, orientation fixation methods reported in the past are inconvenient due to the necessity of chemical modification prior to antibody immobilization
On the other hand, random covalent immobilization may sometimes be ineffective for some commercial therapeutic antibodies, because the immobilized group is likely to be near the binding site of the antibody, and these antibodies are more likely to be on the surface. are immobilized at multiple points, resulting in antibody inactivation

Method used

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  • A Protein Immobilization Method Based on Surface Plasmon Resonance Imaging Biosensor Chip
  • A Protein Immobilization Method Based on Surface Plasmon Resonance Imaging Biosensor Chip
  • A Protein Immobilization Method Based on Surface Plasmon Resonance Imaging Biosensor Chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Immobilization of the therapeutic antibody hR3 on the two-dimensional surface of the chip

[0078] According to the above model, the optimal dilution factor D is obtained F = 1000, the therapeutic antibody hR3 was immobilized on the two-dimensional surface of the chip and detected by surface plasmon resonance imaging according to the following steps:

[0079] (1) Alternately clean the chip with ethanol and deionized water, dry it with nitrogen gas; put it into a plasma cleaner for cleaning for 3 minutes;

[0080] (2) Prepare the mixed thiol solution required for the self-assembled monolayer, mix 1mM HS-PEG 6 -COOH ethanol solution with 1 mM HS-PEG 4 -OCH 3 Ethanol solution diluted 1000 times;

[0081] (3) Immerse the chip in the prepared mixed thiol solution, and incubate overnight (12 hours) at a constant temperature at 4°C. Take out the chip, wash the surface of the chip alternately with ethanol and deionized water, and dry it with nitrogen gas for l...

Embodiment 2

[0086] Example 2: Covalent immobilization of therapeutic antibodies on a three-dimensional surface of a chip

[0087] The three-dimensional surfaces based on surface growth are all developed on the basis of two-dimensional surfaces, and the methods are to fix initiators on the two-dimensional surfaces first, and then carry out polymerization growth. A typical example is the surface self-assembly of monothiol-terminated hyperbranched polyethylene glycol structures (HPGs). These surfaces have been used in surface plasmon resonance imaging, especially in the detection of protein arrays and small molecule microarrays. In some cases, for larger proteins such as antibodies, the steric hindrance on the surface will directly affect the detection function of the surface. If the initiator density on the surface is too high, the distance between the hyperbranched polyethylene glycol molecules will be too short in the vertical space, resulting in considerable steric hindrance. When a ...

Embodiment 3

[0099] Example 3: Immobilization of antibodies with His tag on the chip

[0100] Because many proteins have been purified using NTA / Ni 2+ In the column method, the protein usually has a His tag. In some studies, such as the identification of total biomarkers in cell lysates, quantitative binding information is important, and NTA surfaces are a good choice for the immobilization of such proteins. For the necessary use of Ni 2+ ions, it will cause non-specific adsorption on the surface, so, under the premise of ensuring the amount of protein immobilization, Ni 2+ The less the content of the better, this translates into a problem of controlling the amount of NTA on the surface.

[0101] For His-tagged antibodies, in the covalent immobilization method, the ideal state is that one NTA binds a His-tagged protein, too much will only increase non-specific adsorption. As previously discussed, the average length of a monoclonal antibody is 15nm, corresponding to an optimal dilutio...

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Abstract

The present invention provides a protein immobilization method based on a surface plasmon resonance imaging biosensor chip. The method establishes a model for protein immobilization, optimizes the chemical modification of the chip surface according to the model, and controls the distance between active sites, thereby Ability to immobilize proteins on surface plasmon resonance imaging biosensor chips. The method of the present invention ensures the single-point immobilization of the protein, thus avoiding the inactivation caused by the multi-point immobilization of the protein, and it is confirmed by the surface plasmon resonance imaging technique that the protein immobilized by this method can obtain an optimized detection signal.

Description

technical field [0001] The invention belongs to the field of protein chip arrays, and relates to a protein immobilization method based on a surface plasmon resonance imaging biosensor chip. Background technique [0002] In the field of immunoassays, directed arrays of coated antibodies (primary antibodies) have attracted widespread interest and attention. In traditional detection, the coated antibody is passively adsorbed on the surface of the substrate (usually a thermoplastic polymer, such as polystyrene), so that only 20-30% of the coated antibody obtains the desired orientation and has detection activity. In typical immunoassays, little attention has been paid to the problem of inefficient immobilization of coated antibodies. Coating antibodies are usually immobilized on the solid surface in excess to ensure that enough antibodies are adsorbed on the surface with the proper orientation to generate a reliable detection signal. However, when extended to micro- or nano-le...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N21/552
Inventor 朱劲松哈维尔·巴蒂斯塔·佩雷斯杨墨迪彭德拉·特亚吉埃内斯托·莫雷诺洛尼·卡尔沃程志强李少鹏周文菲
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA