Microfluidic immune imprinting chip and preparation method and application thereof

A technology of immunoblotting and microfluidics, which is applied in the direction of material inspection products, measuring devices, instruments, etc., can solve the problems of high experimental cost, inability to detect protein molecular weight, expensive antibodies, etc., and achieve the effect of simple operation

Active Publication Date: 2011-06-08
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • 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 blot experiment requires a lot of antibody solution (hundreds of microliters to several milliliters), and the antibody is expensive, which makes the western blot experiment more costly
However, the above-mentioned microfluidic immunoassay cannot detect the molecular weight of the protein, and the molecular weight of the protein as an important information can be used to eliminate false positive signals and identify the post-translational modification of the protein

Method used

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  • Microfluidic immune imprinting chip and preparation method and application thereof
  • Microfluidic immune imprinting chip and preparation method and application thereof
  • Microfluidic immune imprinting chip and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Microfluidic Western Blotting Method

[0049] This example is the microfluidic immunoblotting method provided by the present invention, the schematic diagram is shown in figure 1 , the specific operation steps are detailed as follows.

[0050] 1. Preparation of microfluidic chip template

[0051] 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.

[0052] 2. Preparation of Microfluidic Pipelines

[0053] The preparation method is soft etching technology, and the preparation material is polydimethylsiloxane (PDMS, polydimethylsiloxane), which is a transparent and viscous liquid in a normal state. After reacting w...

Embodiment 2

[0086] Example 2 Sensitivity of Microfluidic Western Blotting Methods

[0087] In western blot experiments, sometimes the expression level of the protein to be detected in cells is very low, so the detection sensitivity is an important indicator of western blot experiments. This example compares the detection sensitivity of the microfluidic western blotting method provided by the present invention and the traditional western blotting method.

[0088] The whole protein of NIH-3T3 cells was loaded in different sample wells of polyacrylamide gel in a 2-fold gradient dilution. For the Annexin II protein on two PVDF membranes under the same loading and transfer conditions, the traditional western blotting method and the microfluidic western blotting method were used to compare the sensitivity detection. The results are as follows: figure 2 shown.

[0089] For the specific steps of the microfluidic immunoblotting method, refer to Example 1, wherein the primary antibody used is ...

Embodiment 3

[0093] Example 3 Linearity Analysis of Microfluidic Western Blotting Methods

[0094] The traditional western blotting method can be used for semi-quantitative analysis of proteins, and the detection signal will increase with the increase of the amount of target protein on the membrane. In this example, the linearity analysis between the microfluidic western blotting method provided by the present invention and the traditional western blotting method was carried out to determine whether the method provided by the present invention is suitable for semi-quantitative analysis of proteins.

[0095] In different sample wells of polyacrylamide gel (the width of the sample well is 5.08mm, and the thickness is 0.75mm). The Annexin II protein on the PVDF membrane under the same sample loading and membrane transfer conditions was detected by traditional western blotting method and microfluidic western blotting method, and the fluorescent signal was plotted against the protein loading ...

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Abstract

The invention provides a microfluidic immune imprinting chip and a preparation method and application thereof. The provided microfluidic immune imprinting chip comprises: 1) a substrate fixed with protein strips separated according to molecular weight; and 2) a microfluidic pipeline. The invention also provides a method for the microfluidic immune imprinting chip, and application of the microfluidic immune imprinting chip to detecting various proteins in a sample, detecting molecular weight markers of the proteins, and optimizing antibody concentration in an immune reaction. Various proteins in a biological sample can be detected through a single immune imprinting experiment only, and the detection sensitivity is high; moreover, the molecular weights of the proteins can be detected, the operation is simple, the immune detection efficiency can be effectively improved, and the cost is reduced.

Description

technical field [0001] The invention relates to a microfluidic immunoblotting chip, its preparation method and application. 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 micropipes 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 growth. [0003] Immunoblotting, also known as Western blotting, is an effective method for identifying a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/535
Inventor 潘文颖张伟蒋兴宇
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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