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Reversible fixture for aglucon of streptavidin envelope chip surface

A ligand and chip technology, applied in SA-chip detection system, realizes the field of reversible immobilization of ligands on the surface of SA-chip, can solve the problem of high cost of application experiments, and achieve the effect of reversible immobilization

Inactive Publication Date: 2007-07-18
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the strong interaction between SA and biotin, the SA-chip becomes a one-time commodity, and the experimental cost is extremely expensive for applications that need to study the properties of different ligands and often need to change the fixed amount of ligands.

Method used

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  • Reversible fixture for aglucon of streptavidin envelope chip surface
  • Reversible fixture for aglucon of streptavidin envelope chip surface
  • Reversible fixture for aglucon of streptavidin envelope chip surface

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1. Immobilization and regeneration of ScFV-Z186-SBP ligand on the surface of SA chip

[0115] The present invention uses a BIAcore biosensor system (BIAcore3000), and operates according to the instructions for use. After the SA chip was locked in the instrument, it was first injected with 50 mM NaOH (containing 1 M NaCl) three times, each time for 1 min, with a flow rate of 40 μL / min. ScFV-Zl86-SBP was dissolved in running buffer (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.005% Surfactant P20), with a final concentration of 20μg / mL. After the baseline of the SPR (Surface Plasmon Resonance) response graph (sensorgram) was stable, the ligand solution was injected at a flow rate of 5 μL / min for 7 minutes. To determine the maximum amount of binding, three consecutive injections were performed. After the first injection, there was a response of approximately 2700 RU (5 μL / min, 7 min). After continuous injection, the response value rose to 3300RU and basically rea...

Embodiment 2

[0118] Example 2. Immobilization and regeneration of Nano-label modified short peptides on the surface of SA chip

[0119] Nano-label modified short peptides were dissolved in running buffer with a final concentration of 200μg / mL. After the baseline of the SPR response graph (sensorgram) was stable, the ligand solution was injected at a flow rate of 5 μL / min for 4 minutes. To determine the maximum amount of binding, six consecutive injections were made.

[0120] The molecular weight of the nano-tag modified short peptide used in this experiment is 2.2 KD, because the lower limit of molecular weight that can be detected by BIAcore3000 is 200 D, which indicates that the response value caused by this short peptide will be lower when immobilized, see Figure 5. After six consecutive injections, saturated binding was basically achieved, with a response value of 136 RU.

[0121] Carry out the regeneration process with the completely similar method of embodiment 1. Because the affi...

Embodiment 3

[0122] Example 3. Binding kinetics experiment of ScFV-Z186-SBP and its antigen

[0123] In order to verify the feasibility of the immobilization method established in this paper, the kinetic binding process of scFv-Z186-SBP and its antigen was analyzed on the same chip (SA-chip). ScFV-Z186-SBP was dissolved in running buffer with a final concentration of 20 μg / mL. The concentration gradient of the antigen ranged from 1430, 715, 358, 179, 90 and OnM. After the baseline of the SPR response graph (sensorgram) was stable, the ScFV-Z186-SBP solution was injected at a flow rate of 5 μL / min for 7 minutes. Considering the weak baseline drift effect, the sensorgram formed by the blank control was subtracted from five concentration gradients (1430, 715, 358, 179, 90 and OnM). After the injection of each concentration of antigen, wash with running buffer for 30 min to allow the natural dissociation of the bound antigen. All tests were performed at room temperature with a flow rate of ...

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Abstract

The present invention discloses one kind of ligand fixing mode for the streptavidin coated chip (SA-chip) surface in BIAcore biosensor. In traditional SA-chip, the ligand is fixed by means of the specific mutual action between biotin and streptavidin, and the very powerful mutual action makes the SA-chip become disposable commodity. Instead, the present invention fuses the affinity label and the target ligand by means of the mutual action between streptavidin and affinity label to realize the directional fixing of target ligand. Under mild controllable condition, the chip surface may be regenerated for reuse of the SA-chip. The new ligand fixing process results in greatly lowered experiment cost and possesses high application value.

Description

technical field [0001] This technology relates to the field of biosensors and biotechnology, more specifically, the present invention relates to a method for realizing the reversible immobilization of ligands on the surface of SA-chip, and a method for realizing the reversible immobilization of ligands on the surface of SA-chip Fixed SA-chip detection system. Background technique [0002] The BIAcore biosensor system is an instrument provided by Amersya for the analysis of biomolecular interactions. The system is mainly composed of three parts: an optical detection system, a sample delivery system, and an output system, as shown in Figure 1. The BIAcore system provides various types of chips for the immobilization of different ligands. Carboxymethyldextran-coated chips (CM chips) are the most versatile chip, suitable for most proteins, but because they are immobilized by amino groups on the surface of proteins, they form a heterogeneous surface, and some Ligands lose biolo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/543
Inventor 张先恩李永进毕利军周亚凤张吉斌陈媛媛李炜张治平
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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