Method for fixing protein on surface of polydimethylsiloxane

A polydimethylsiloxane and surface immobilization technology, which is applied in material inspection products, measuring devices, instruments, etc., can solve the problems of complex protein types and quantities, unstable operation, poor specificity, etc., and achieves good specificity, The effect of improving sensitivity and increasing protein quality

Inactive Publication Date: 2011-11-16
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The object of the present invention is to provide a method for immobilizing proteins on the surface of PDMS to overcome the defects of existing methods for immobilizing proteins on the surface of PDMS or poor specificity or instability or complex operation or limited protein types and quantities

Method used

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  • Method for fixing protein on surface of polydimethylsiloxane
  • Method for fixing protein on surface of polydimethylsiloxane
  • Method for fixing protein on surface of polydimethylsiloxane

Examples

Experimental program
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Effect test

Embodiment 1

[0035] In this example, the low-density lipoprotein apoB 100 antibody mAb LDL-10 / 17 (purchased from Abcam (Cambridge, MA, USA)) was used as the protein to be immobilized, and the protein to be immobilized was immobilized on the PDMS surface according to the following method, immobilized The schematic diagram of the protein is as figure 1 Shown:

[0036] (1) Form a layer of biotinylated PDMS thin film on the surface of 60mm×60mm PDMS support layer. That is, add 10 microliters of 5 mg / mL phospholipid biotin in chloroform solution to the PDMS prepolymer / n-hexane (mass ratio is 1:2, and the quality of the PDMS prepolymer / n-hexane solution is 1 g), and then coat the The surface of the PDMS support layer was cured at 80°C for 20 minutes to form a layer of biotinylated PDMS film.

[0037] (2) In order to prevent the non-specific adsorption of protein on the surface of biotinylated PDMS film, use 10mmol / L pH 7.4 phosphate buffer solution (DPBS) containing 0.1wt% β-D-dodecyl-N-maltos...

Embodiment 2

[0043] In this example, alpha-fetoprotein antibody (purchased from American Research Products (Belmont, MA, USA)) was used as the protein to be immobilized.

[0044] (1) Form a layer of biotinylated PDMS thin film on the surface of 60mm×60mm PDMS support layer. That is, add 2 microliters of 5 mg / mL phospholipid biotin in chloroform solution to the PDMS prepolymer / n-hexane (mass ratio is 1:2, the mass ratio of PDMS prepolymer / n-hexane is 1 g), and then coat the PDMS The surface of the support layer was cured at 80°C for 20 minutes to form a layer of biotinylated PDMS film.

[0045] (2) In order to prevent non-specific adsorption of proteins on the surface of biotinylated PDMS, soak with 10mmol / L pH 7.4 phosphate buffered saline solution (DPBS) containing 0.2wt% β-D-dodecyl-N-maltose Biotinylate the PDMS surface for 5 min.

[0046] (3) In order to specifically bind streptavidin to the biotin group, soak the biotinylated PDMS surface with 200 μg / mL streptavidin in DPBS for 30 m...

Embodiment 3

[0051] In this example, the intrinsic PDMS film and the modified PDMS film in Example 1 were characterized by FTIR / R760 infrared spectrometer (equipped with ATR components) ( Figure 2A-2B ). The spectrum (curve 1) of the intrinsic PDMS film is at 3386cm -1 There is no absorption peak nearby, and the spectrum (curve 2) of the PDMS thin film treated by DPBS solution is at 3386cm -1 There is an obvious broad absorption peak nearby, which indicates that the nonionic surfactant β-D-dodecyl-N-maltose molecule with many hydroxyl groups is adsorbed onto the PDMS surface. Compared with the PDMS film treated with DPBS solution, the spectrum (curve 3) of the PDMS film treated with the DPBS solution containing streptavidin is at 3386cm -1 There were no obvious changes nearby, which indicated that the surfactant β-D-dodecyl-N-maltose effectively prevented the non-specific adsorption of streptavidin on the PDMS surface. However, when the biotinylated PDMS film was modified with streptav...

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Abstract

The invention provides a method for fixing a protein on the surface of polydimethylsiloxane. The method is characterized in that a biotinylated polydimethylsiloxane film is prepared on the solidified surface of polydimethylsiloxane, a protein is fixed on the surface of polydimethylsiloxane by chemical technologies of biotin-avidin and water-soluble carbodiimide and a technology of 3-mercaptopropionic acid stabilized gold nano-particles, and beta-D-dodecyl-N-maltose and bovine serum albumin are utilized respectively for blockading the non-specific adsorption of a protein on the surface of polydimethylsiloxane and activated residual carboxylic groups on the surface of a 3-mercaptopropionic acid stabilized gold nano-particle. In the invention, the surface of a 3-mercaptopropionic acid stabilized gold nano-particle contains multiple carboxylic groups which can combine molecules of multiple proteins needing to be fixed thus multiple proteins needing to be fixed can be fixed on the surface of polydimethylsiloxane and a sensitivity of a subsequent enzyme-linked immuno sorbent assay (ELISA) is improved effectively.

Description

technical field [0001] The invention belongs to the technical field of surface chemical modification, in particular to a method for immobilizing proteins on the surface of polydimethylsiloxane. Background technique [0002] Polydimethylsiloxane (PDMS), commonly known as silicone rubber, is a polymer material with very good biocompatibility and is currently one of the most widely used materials for microfluidic chip processing. The PDMS microfluidic chip has the following advantages: (1) The production cost is low, the production process is convenient and fast, and it can be mass-produced; (2) The monomer can be polymerized at low temperature, and there is no special requirement for the equipment; (3) The surface energy is low, and it is easy to integrate with PDMS And other materials such as glass, other polymer materials for temporary or permanent sealing, and in the process of use, if the micro-channel is blocked, the temporarily sealed chip can be disassembled and cleaned...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531
Inventor 吴会灵陈肇娜纪季范国荣刘宝红杨芃原
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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