阅读说明：本技术 一种微流控芯片及其制备方法 (Micro-fluidic chip and preparation method thereof ) 是由 王云兵 李高参 马博轩 杨立 邹耀中 苏云鹏 江鹏 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种微流控芯片及其制备方法。芯片包括经过疏水改性的芯片基底,芯片基底上覆有磷脂膜,磷脂膜上镶嵌有经过核苷酸修饰物修饰后的跨膜蛋白。经疏水改性的芯片基底在电解质溶液浸润后,可以实现与磷脂膜的稳定结合；经稳定化处理的两性磷脂分子具有更高的成膜率,其在芯片基底形成磷脂膜后,具有更好的稳定性,可实现更长时间的测序工作；经核苷酸修饰的跨膜蛋白可嵌入磷脂膜,作为生物纳米孔实现基因测序工作,经过核苷酸化修饰,其在基因测序的效率及准确性上都有较大的提升。采用本发明所述的生物纳米孔微流芯片系统,可大大提升自动化基因测序速度,测序准确性并有效降低测序成本。(The invention discloses a micro-fluidic chip and a preparation method thereof. The chip comprises a chip substrate which is subjected to hydrophobic modification, wherein a phospholipid membrane is covered on the chip substrate, and transmembrane protein modified by a nucleotide modifier is inlaid on the phospholipid membrane. After the chip substrate subjected to hydrophobic modification is soaked in an electrolyte solution, stable combination with a phospholipid membrane can be realized; the amphiprotic phospholipid molecules subjected to the stabilizing treatment have higher film forming rate, have better stability after a phospholipid film is formed on a chip substrate, and can realize longer-time sequencing work; the transmembrane protein modified by nucleotide can be embedded into a phospholipid membrane to be used as a biological nanopore to realize gene sequencing work, and the efficiency and the accuracy of gene sequencing are greatly improved through nucleotide modification. The biological nanopore microfluidic chip system can greatly improve the automatic gene sequencing speed, improve the sequencing accuracy and effectively reduce the sequencing cost.)

1. A microfluidic chip, characterized in that: the chip comprises a chip substrate which is subjected to hydrophobic modification, wherein a phospholipid membrane is covered on the chip substrate, and transmembrane protein modified by a nucleotide modifier is inlaid on the phospholipid membrane.

2. The microfluidic chip of claim 1, wherein: the transmembrane protein is alpha-HL, Msp A or phi 29; the nucleotide modifier is a compound of a hydrophilic polymer and nucleotide, and the hydrophilic polymer and the nucleotide in the compound are connected through a covalent bond.

3. The microfluidic chip of claim 2, wherein: the hydrophilic polymer is polylysine, polyglutamic acid or polyethylene glycol; the nucleotide is adenine nucleotide, thymine nucleotide, cytosine nucleotide or guanine nucleotide.

4. The method for preparing the microfluidic chip according to any one of claims 1 to 3, comprising the steps of:

s1: carrying out hydrophobic modification on a chip substrate;

s2: forming a phospholipid membrane on the chip substrate subjected to hydrophobic modification;

s3: modifying and modifying transmembrane protein by adopting a nucleotide modifier;

s4: embedding the modified transmembrane protein on a phospholipid membrane to complete the preparation of the microfluidic chip.

5. The method of claim 4, wherein the hydrophobic modification of the chip substrate comprises the steps of:

SS 1: pretreating the surface of the substrate by Su-8;

SS 2: carrying out epoxy ring opening on the pretreated substrate;

SS 3: grafting perfluorinated compounds on the substrate after the ring opening of the epoxy to obtain a hydrophobic modified chip substrate;

the substrate material is polystyrene or polycarbonate.

6. The method of claim 5, wherein: the perfluoro compound is at least one of perfluorodecylethylene, perfluoroundecanoic acid, heptadecafluoroundecanoyl chloride, perfluorododecylamine, perfluorodecyltrimethoxysilane and perfluorodecyldimethylchlorosilane.

7. The method according to claim 4, wherein the formation of the phospholipid film in S2 comprises the steps of:

SS 1: mixing phospholipid molecules, hydrophobic monomers and photoinitiators according to a molar ratio of 1: 0.5-3 to obtain stabilized amphiprotic phospholipid molecules;

SS 2: dissolving the stabilized amphiprotic phospholipid molecules in an electrolyte solution to obtain an amphiprotic phospholipid molecule solution;

SS 3: soaking the hydrophobically modified chip substrate in an amphiprotic phospholipid molecular solution, and then carrying out ultraviolet irradiation for 10-60 min to obtain the hydrophobic modified chip substrate.

8. The method of claim 7, wherein: the molar ratio of phospholipid molecules, hydrophobic monomers and photoinitiators in the stabilized ampholytic phospholipid molecules is 1:1: 1.

9. The production method according to claim 7 or 8, wherein: the phospholipid molecules are diphytanoyl phosphatidylcholine and/or diphytanoyl phosphatidylethanolamine; the hydrophobic monomer is n-butyl methacrylate and/or ethylene glycol dimethacrylate; the photoinitiator is 2, 2-diethoxy-1-phenylhexanone and/or 2-hydroxy-2-methyl-1-phenyl acetone.

10. The method of claim 4, wherein: the modified transmembrane protein is embedded on the phospholipid membrane in a self-assembly mode.

Technical Field

The invention belongs to the technical field of biological science, and particularly relates to a micro-fluidic chip and a preparation method thereof.

Background

In recent years, with the introduction of the concept of "precision medicine" and the arrival of the age of big data, the sequencing work of human genes has gained more and more importance in the process of improving the national health level. Among them, the biological nanopore gene sequencing technology gains wide attention due to its good sequencing accuracy, high-efficiency sequencing speed and low sequencing cost.

As the core of the biological nanopore gene sequencing technology, a microfluidic chip system plays a crucial role in the research and development of the nanopore sequencing technology. However, the development of this type of chip is still not mature, and there is still a need for improvement in terms of accuracy, stability and lifetime to meet the requirements of clinical tests. Therefore, how to improve the stability, accuracy and service life of the microfluidic chip system has become one of the important factors influencing the further application of the microfluidic chip system in the biomedical industry.

Disclosure of Invention

Aiming at the prior art, the invention provides a microfluidic chip and a preparation method thereof, aiming at solving the problems of non-ideal stability, accuracy and service life of the existing microfluidic chip.

In order to achieve the purpose, the invention adopts the technical scheme that: the microfluidic chip comprises a chip substrate subjected to hydrophobic modification, wherein a phospholipid membrane is coated on the chip substrate, and transmembrane protein modified by a nucleotide modifier is inlaid on the phospholipid membrane.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the transmembrane protein is alpha-HL, Msp A or phi 29; the nucleotide modifier is a compound of a hydrophilic polymer and nucleotide, and the hydrophilic polymer and the nucleotide are connected through a covalent bond.

The sensitivity and accuracy of gene sequencing can be improved by carrying out nucleotide modification on the transmembrane protein. Covalent modification of transmembrane proteins by polymer-nucleotide complexes is achieved by modifying functional groups such as biotin, which are reactive with proteins, at the end groups of hydrophilic polymers.

Further, the hydrophilic polymer is polylysine, polyglutamic acid or polyethylene glycol; the nucleotide is adenine nucleotide, thymine nucleotide, cytosine nucleotide or guanine nucleotide.

The invention also claims a preparation method of the microfluidic chip, which comprises the following steps:

s1: carrying out hydrophobic modification on a chip substrate;

s2: forming a phospholipid membrane on the chip substrate subjected to hydrophobic modification;

s3: modifying and modifying transmembrane protein by adopting a nucleotide modifier;

s4: embedding the modified transmembrane protein on a phospholipid membrane to complete the preparation of the microfluidic chip.

On the basis of the technical scheme, the preparation method can be further improved as follows.

Further, the hydrophobic modification of the chip substrate comprises the following steps:

SS 1: pretreating the surface of the substrate by Su-8;

SS 2: carrying out epoxy ring opening on the pretreated substrate;

SS 3: grafting perfluorinated compounds on the substrate after the ring opening of the epoxy to obtain a hydrophobic modified chip substrate;

the material of the substrate is polystyrene or polycarbonate.

The epoxy ring opening of the substrate can be realized by soaking the pretreated substrate in amino ethanol, the epoxy ring opening is realized by the reaction of the substrate and the amino ethanol, so that a hydroxyl functional group is introduced to the surface of the substrate, the hydroxyl group and a functional group on a perfluorinated compound are subjected to chemical reaction, and a perfluorinated compound is grafted on the substrate to obtain the hydrophobic modified substrate. The perfluorinated compound modified chip substrate can better stabilize the phospholipid membrane and realize long-acting and stable gene sequencing.

Further, the perfluoro compound is at least one of perfluorodecylethylene, perfluoroundecanoic acid, heptadecafluoroundecanoyl chloride, perfluorododecylamine, perfluorodecyltrimethoxysilane and perfluorodecyldimethylchlorosilane.

Further, the formation of the phospholipid film in S2 includes the steps of:

SS 1: mixing phospholipid molecules, hydrophobic monomers and photoinitiators according to a molar ratio of 1: 0.5-3 to obtain stabilized amphiprotic phospholipid molecules;

SS 2: dissolving the stabilized amphiprotic phospholipid molecules in an electrolyte solution to obtain an amphiprotic phospholipid molecule solution;

SS 3: soaking the hydrophobically modified chip substrate in an amphiprotic phospholipid molecular solution, and then carrying out ultraviolet irradiation for 10-60 min to obtain the hydrophobic modified chip substrate.

Further, the molar ratio of phospholipid molecules, hydrophobic monomers and photoinitiators in the stabilized ampholytic phospholipid molecules is 1:1: 1.

Further, the phospholipid molecule is diphytanoyl phosphatidylcholine or diphytanoyl phosphatidylethanolamine; the hydrophobic monomer is n-butyl methacrylate or ethylene glycol dimethacrylate; the photoinitiator is 2, 2-diethoxy-1-phenylhexanone or 2-hydroxy-2-methyl-1-phenyl acetone.

Furthermore, the modified transmembrane protein is embedded on the phospholipid membrane in a self-assembly mode.

Transmembrane protein can spontaneously form an assembly structure in an electrolyte solution, so that the microfluidic chip disclosed by the invention is simple and convenient in preparation method, and a microfluidic chip with excellent performance can be quickly obtained.

The invention has the beneficial effects that: the invention creatively improves the aspects of the chip substrate, the phospholipid molecules and the transmembrane protein, and improves the binding force of the chip substrate to the phospholipid membrane, the membrane forming stability of the phospholipid membrane and the recognition capability of the transmembrane protein nanopore to the gene sequence, thereby realizing more accurate, stable and durable gene sequencing work.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.