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A method for protein crystallization by changing the roughness of crystallization substrates using soft lithography chips

A protein crystallization and roughness technology, which is applied to the preparation method of peptides, chemical instruments and methods, and photolithography on patterned surfaces, etc., can solve the problem of concentration difficulty reaching supersaturation, crystal nuclei cannot be formed, low success rate of crystallization, etc. problem, to achieve the effect of increasing the success rate of screening, increasing the probability of protein nucleation, and improving the success rate of protein crystallization

Inactive Publication Date: 2019-09-24
NORTHWESTERN POLYTECHNICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For some precious protein samples, the concentration is difficult to reach supersaturation, and crystal nuclei cannot be formed, which directly leads to the problem of low crystallization success rate.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Screening of proteinase K crystallization conditions

[0034]The first step: PDMS chip design and production: (1) PDMS chip design: design a series of micro-pillar arrays closely arranged on the PDMS chip interface, the diameter of the micro-pillars is 5 μm, the height of the micro-pillars is 10 μm, and the The spacing is 100 μm; (2) Chip and template preparation: a) Prepare the substrate: use 98% concentrated sulfuric acid:hydrogen peroxide according to the volume ratio of 3:1 to clean the single-polished silicon wafer, and clean the silicon wafer with ultra-pure Rinse with water, soak in absolute ethanol and acetone for 1 min, then rinse with ultrapure water, and dry the water on the silicon wafer at 200°C to obtain a substrate for photoetching; b) Spreading: pour the negative photoresist On the substrate, it is coated with a film applicator to form a uniform thin layer of photoresist, and substrates of different thicknesses are prepared for photolithograph...

Embodiment 2

[0039] Embodiment 2: Screening of lysozyme protein crystallization conditions

[0040] The first step: PDMS chip design and production: (1) PDMS chip design: design micro-column arrays on the PDMS chip interface to be closely arranged in a region with a length of 1mm and a width of 0.5mm, and the distance between the arrays is 1mm, where The diameter of the microcolumn is 50 μm, the height of the microcolumn is 10 μm, and the spacing of the microcolumn is 100 μm; (2) Chip and template preparation: a) Prepare the substrate: use 98% concentrated sulfuric acid: hydrogen peroxide according to the volume ratio of 3:1 The prepared lotion was used to clean single-polished silicon wafers. The cleaned silicon wafers were rinsed with ultra-pure water, soaked in absolute ethanol and acetone for 1 min in sequence, then rinsed with ultra-pure water, and dried at 200°C to obtain Substrates for photoetching; b) Spreading: Pour the negative photoresist on the substrate and apply it with a fil...

Embodiment 3

[0045] Embodiment 3: the screening of ribonuclease A crystallization condition

[0046] The first step: PDMS chip design and production: (1) PDMS chip design: design a series of microcolumn arrays on the PDMS chip interface, and each group of microcolumn arrays is closely arranged in an area with a length of 1cm and a width of 1mm. The distance between them is 1 mm, the diameter of the microcolumns is 30 μm, the height of the microcolumns is 100 μm, and the distance between the microcolumns is 20 μm; (2) chip and template preparation: a) prepare the substrate: use 98% concentrated sulfuric acid: hydrogen peroxide according to the volume Clean single-polished silicon wafers with a lotion ratio of 3:1, rinse the cleaned silicon wafers with ultra-pure water, soak in absolute ethanol and acetone for 1 min, then rinse with ultra-pure water, and dry the silicon wafers at 200 °C. Moisture on the sheet to obtain a substrate for photoetching; b) Spreading: Pour the negative photoresist...

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Abstract

The invention provides a method for changing the roughness of a crystalline basement by using a soft lithography chip for protein crystallization. The method comprises the following steps: firstly, designing and producing a PDMS (Polydimethylsiloxane) chip meeting the crystallization requirements of protein; secondly, dropwise adding a protein crystallization solution on the surface of the PDMS chip, standing and crystallizing. According to the method provided by the invention, by designing the PDMS chip, a controllable crystallization interface with uniform roughness is realized, so that the nucleation probability of the protein on the surface of the basement is improved, and further the aim of improving the success rate of protein crystallization is achieved. According to the method, the screening success rate of protein crystallization conditions can be improved by 1 to 5 times.

Description

technical field [0001] The invention relates to a method for modifying a protein crystallization substrate by using soft-etching chip technology, in particular to a method for improving the success rate of protein crystallization by changing the crystal interface roughness by using soft-etching technology. Background technique [0002] Protein is the executor of life activities, and its structure is the premise and basis of functional research. Therefore, the analysis of protein structure is of great significance to elucidate its biological function. At present, more than 88% of the structures in the protein structure database are obtained by X-ray diffraction technology, and high-quality protein crystals are the premise and basis for obtaining structures by X-ray diffraction technology. At present, the low success rate of screening protein crystallization conditions is an important bottleneck restricting the development of X-ray diffraction technology. Therefore, how to imp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/30G03F7/00
Inventor 张辰艳杨雪舟尹大川董晨闫二开
Owner NORTHWESTERN POLYTECHNICAL UNIV
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