Multiphase microfluidic immunoblotting chip, and preparation method and application thereof

A technology of immunoblotting and microfluidics, which is applied in the fields of biotechnology and tissue engineering, can solve the problems of difficult biological laboratory use, high cost, and unsatisfactory detection, and achieve the effects of reducing costs, saving time, and improving effects and quality

Active Publication Date: 2013-07-03
BEIJING NANO ACE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing problems of immunoblotting are: (1) one experiment can only detect the expression of a protein in a biological sample
It cannot meet the purpose of simultaneous detection of multiple proteins in cells or tissues in biological research
(2) A Western blotting experiment requires a large amount of antibody solution (hundreds of microliters to several milliliters), and the antibody is expensive, which makes the cost of Western blotting higher.
However, the number of proteins that can be detected by multicolor fluorescein labeling and multicolor quantum dot labeling methods is limited, because the wavelength ranges of excitation light and emission light of different labeled substances (fluorescein or quantum dots) will overlap. The number of proteins detected is mostly less than 5
The disadvantage of surface-enhanced Raman immunoblotting is that the spectral profiles are complex and difficult to distinguish when detecting mixtures of multiple proteins
[0006] There are also some reports to use microfluidic technology to detect various proteins formed by electrophoresis, for example, the whole electrophoresis and transfer process are concentrated in a tiny channel (He, M., Herr, A.E., Nat.Prot.2010, 5 , 1844-1856), but the problem with this technology is that it cannot provide information on the molecular weight of the target protein, and the readout method is too complicated to be used in general biological laboratories; another example, we invented a system before, using parallel The microfluidic channels for introducing antibodies to detect target proteins (Pan, W.Y.; Chen, W.; Jiang, X.Y.; Anal. Chem. 2010, 82, 3974-3976) can solve the above problems, but this method can only Processing one sample obviously cannot meet the needs of processing many samples at the same time in biological experiments

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  • Multiphase microfluidic immunoblotting chip, and preparation method and application thereof
  • Multiphase microfluidic immunoblotting chip, and preparation method and application thereof
  • Multiphase microfluidic immunoblotting chip, and preparation method and application thereof

Examples

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Embodiment 1

[0045] This example illustrates the preparation method of the multiphase microfluidic immunoblotting chip of the present invention

[0046] 1. Preparation of microfluidic chip template

[0047] The main process of preparation is photolithography, which is to make a photoresist silicon wafer template that is completely consistent with the pattern on the designed mask by using the characteristic that the photoresist can change its properties under ultraviolet irradiation. The specific preparation method can be found in Y Xia, G. Whitesides, Annual Review of Materials Science 1998, 28, 15.

[0048] 2. Preparation of sheet with multiple microfluidic grooves

[0049] The preparation method is soft etching technology, and the preparation material is polydimethylsiloxane (PDMS, poly-dimethylsiloxane), which is a transparent and viscous liquid in a normal state. After being mixed with a curing agent (184 silicone elastomer curing agent , purchased from Dow Corning Corporation of the...

Embodiment 2

[0081] This example is used to illustrate the multiphase detection capability of the multiphase microfluidic immunoblotting chip of the present invention. The implementation steps are as follows:

[0082] As described in Example 1, load 80 μg of lysed whole protein of NIH 3T3 cells into two swimming lanes, perform electrophoresis according to the preferred time, transfer to membrane, and then dry the membrane loaded with protein, use PDMS monomer and The curing agent was prepared in a mass ratio of 20:1, and baked at 80° C. for 25 minutes to prepare a PDMS chip, and the film and the chip were bonded together by light pressure. From left to right, different antibodies are passed into the channels, specifically, 1 and 2: the mixture of anti-annexin antibody and anti-Pan-14-3-3 antibody; 3 and 4: the passage Mixture of anti-actin antibody (anti-actin antibody) and anti-Pan-14-3-3 antibody; 5 and 6: pass through anti-actin antibody (anti-actin antibody) and anti-membrane Protein...

Embodiment 3

[0084] This example is used to illustrate the ability of the multiphase microfluidic immunoblotting chip of the present invention for semi-quantitative detection.

[0085] As described in Example 1, put 10 μg, 5 μg, 2.5 μg, and 1.25 μg of the lysed whole protein of NIH 3T3 cells in the four lanes, perform electrophoresis according to the preferred time, transfer to the membrane, and then transfer the protein-loaded membrane to Drying, using the mass ratio of PDMS monomer and curing agent at 20:1, drying at 80°C for 25 minutes to prepare a PDMS chip, and combining the film and the chip by light pressure. The anti-annexin antibody from Santa Cruz Company was used at an initial concentration of 1:20, and then the antibody solutions were serially diluted to 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280. These seven concentrations of antibodies were passed into seven microfluidic channels respectively, and the concentration of the secondary antibody was 1:2000. The dot matrix diagra...

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Abstract

The invention provides a multiphase microfluidic immunoblotting chip, and a preparation method and applications thereof. The chip comprises a substrate fixed with a separate protein band, and a chip body, wherein at least one micro fluid concave groove is arranged on one surface of the chip body, the micro fluid concave groove and the a polymer film together form at least one microflow passage for antibody circulation, and a via hole is respectively arranged on each end port, corresponding the microflow passage, on another surface, to form a passage inlet and a passage outlet of the microflow passage. The chip preparation method comprises the following steps of: preparing the substrate fixed with the separate protein band; and assembling the prepared substrate and the clip body. The chip is used for protein detection and for preparing a kit for protein detection. Single-time immunoblotting reaction of the invention can determine existence of multiple target proteins, and the chip and the preparation method has advantages of high detection sensitivity, simple operation, high detection efficiency and low cost.

Description

technical field [0001] The invention relates to a chip, in particular to a multiphase microfluidic immunoblotting chip and its preparation method and application, belonging to the fields of biotechnology and tissue engineering. Background technique [0002] Microfluidics is an analytical technology that uses micron-scale small pipes and structures to finely control microfluids at the microliter level. The corresponding technology carrier is a microfluidic chip. It is based on the soft etching technology derived from the microelectronics process, and builds a microfluidic system by integrating tiny components such as microchannels and microreaction chambers on the chip, loading biological samples or chemical reaction liquids, and using pressure pumps or electroosmotic flow as The power forms a microfluidic path, so that one or more delicate operations or reactions can be performed on the chip to achieve various purposes such as biological detection, chemical synthesis or cell...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543
Inventor 蒋兴宇何沙张翼
Owner BEIJING NANO ACE TECH CO LTD
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