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CN-116559055-B - Microfluidic chip, preparation method, analysis device and analysis method

CN116559055BCN 116559055 BCN116559055 BCN 116559055BCN-116559055-B

Abstract

The application belongs to the technical field of treating microorganisms or particles by electromagnetic sound and other energy, and particularly relates to a microfluidic chip, a preparation method, an analysis device and an analysis method. The micro-fluidic chip comprises a main channel, two or more sample injection channels communicated with the main channel, and a focusing electrode and a deflection electrode are arranged at the bottom of the main channel, wherein the focusing electrode is used for gathering particles, and the deflection electrode is used for directionally moving the particles. According to the micro-fluidic chip, a plurality of sample introduction channels are arranged, so that multiple fluids can enter the main channel at the same time, fluid in the main channel can enter the sample outlet channel or the waste sample channel controllably by adjusting the fluid flow rates of different sample introduction channels, the directional movement of particles is realized, in the moving process, a specific detection point is selected, the specific detection is completed, the dielectric-based online cleaning is realized, the compatibility of various complex samples at the upstream is realized, and the downstream is conveniently subjected to deeper analysis.

Inventors

  • WANG XIXIAN
  • Diao Zhidian
  • MA BO
  • XU JIAN

Assignees

  • 中国科学院青岛生物能源与过程研究所

Dates

Publication Date
20260508
Application Date
20220831
Priority Date
20220127

Claims (6)

  1. 1. A microfluidic chip comprising a main channel (10), characterized in that: two or more sample introduction channels communicated with the main channel (10), A focusing electrode (11) and a deflection electrode (12) are arranged at the bottom of the main channel (10), The focusing electrode (11) is used for gathering particles, The deflection electrode (12) is used for directionally moving particles; when two sample introduction channels are communicated with the main channel (10): The two sample injection channels are a first sample injection channel (13) and a second sample injection channel (14) respectively, and the other end of the main channel (10) is communicated with a sample outlet channel (15) and a waste sample channel (16), wherein the first sample injection channel (13) and the sample outlet channel (15) are arranged on the same side of the main channel (10), and the second sample injection channel (14) and the waste sample channel (16) are arranged on the other side of the main channel (10); through setting two sample introduction channels, buffer solution is introduced into the first sample introduction channel (13) to realize online cleaning of particles (17); when three sample introduction channels are communicated with the main channel (10): The three sample injection channels are respectively a first sample injection channel (13), a second sample injection channel (14) and a third sample injection channel (30), two sample outlet channels and a waste sample channel (16) are communicated at the other end of the main channel (10), the two sample outlet channels are respectively a first sample outlet channel (15) and a second sample outlet channel (31), fluid is introduced into the three sample injection channels at different flow rates, and the outflow channels of the fluid are controlled to be the first sample outlet channel (15), the second sample outlet channel (31) or the waste sample channel (16) through the flow rates; The second sample introduction channel (14) is filled with cell suspension, the third sample introduction channel (30) is filled with buffer solution, the first sample introduction channel (13) is filled with lysis solution, so that the online cleaning and the pyrolysis of the particles (17) are realized, the first sample discharge channel (15) is connected with detection equipment, the lysis solution, water and culture medium flow out through the second sample discharge channel (31), and residual substances of the cell suspension are discharged through the waste sample channel (16); the focusing electrode (11) is a sharp-angle interdigital electrode, and the pointed end of the sharp-angle interdigital electrode points away from the sample introduction channel.
  2. 2. A method for manufacturing a microfluidic chip, which is characterized by comprising the following steps: (1) The focusing electrode (11) and the deflection electrode (12) are fixedly arranged on the basal layer (29), and the main channel (10) and the sample introduction channel are cut on the double faced adhesive tape (28); (2) Bonding one surface of the double-sided tape (28) to a substrate layer (29); (3) And a top cover layer (27) is attached to the other surface of the double-sided adhesive tape (28).
  3. 3. An analysis device, comprising: A particle-carrying capture device for carrying and capturing particles, wherein the particle-carrying capture device is the microfluidic chip according to claim 1; The spectrometer is characterized by comprising a particle carrying and capturing device, a fluid driver, a spectrometer and a light source, wherein the particle carrying and capturing device is used for carrying and capturing particles; the genome analysis device is connected with the particle bearing and capturing device and is used for detecting particle information; And the computer is connected with the spectrometer or the spectrometer and the genome analysis device and is used for controlling the spectrometer and the genome analysis device and recording and analyzing information obtained from the spectrometer and the genome analysis device.
  4. 4. An analysis device according to claim 3, wherein: The micro-fluidic chip is provided with alternating current by a function generator; the spectrum information acquisition end (18) of the spectrometer is aligned with the sharp corner end of the focusing electrode (11); the genomic analysis device is connected to the sample outlet channel (15).
  5. 5. An analysis device according to claim 4, wherein: The sample outlet channel (15) is connected with one end of a post-treatment pipe (22), the other end of the post-treatment pipe (22) is connected with an oil pool (24), a first feeding pipe (23) and a second feeding pipe (25) are communicated with the post-treatment pipe (22), and liquid drops of the oil pool (24) are used for genome analysis.
  6. 6. An analysis method characterized by using a microfluidic chip according to claim 1, comprising the steps of: (1) The connecting device comprises: connecting the function generator with a focusing electrode (11) and a deflection electrode (12) respectively; a spectrum information acquisition end (18) of the spectrometer is aligned with the main channel (10); The computer is respectively connected with the function generator and the spectrometer; (2) Introducing a buffer solution into the main channel (10) through a first sample introduction channel (13), and introducing a fluid containing particles into the main channel (10) through a second sample introduction channel (14); (3) Focusing by applying a voltage to a focusing electrode (11) to cause particles in the fluid to accumulate; (4) Optical detection, namely starting a spectrometer, and transmitting detected signals to a computer program for analysis and processing; (5) Deflection by applying a voltage to the deflection electrode (12) to deflect particles in the fluid; (6) And (3) secondary detection, namely adding the liquid flowing out of the sample outlet channel (15) into enzyme and gel bead (26) suspension containing barcoad, mixing to form liquid drops, and carrying out single-cell sequencing on the liquid drops to realize genome analysis.

Description

Microfluidic chip, preparation method, analysis device and analysis method The invention is an invention application with priority, and the priority basis comprises an invention application with application number of 202210101266.X, patent name of chip, manufacturing method, analysis device method and single cell multi-phenotype analysis application, and application date of 2022, 1 month and 27 days. Technical Field The application belongs to the technical field of treating microorganisms or particles by electromagnetic sound and other energy, and particularly relates to a microfluidic chip, a preparation method, an analysis device and an analysis method. Background Cells are the most fundamental building blocks of the structure and function of most organisms, and research into cells has been focused on. In cell biology, cell populations are classified and studied according to characteristics of cell origin tissue, self morphology, secretion, and the like. The research has great significance on human body drug metabolism, organism behaviors, signal transmission and the like. These studies are based on cell populations, i.e. large numbers of cells, and whether the study of cell morphology or metabonomics is an average of these large numbers of cells. These methods of investigation cannot be used for the investigation of cell differentiation. In recent years, scientists pay more and more attention to single-cell research, and the single-cell research has an increasing effect. Not only relates to drug metabolism and new drug development, but also can well explain the problems of cell molecular mechanism, cell passage and the like. Thus, comprehensive analysis of individual cells is a very important technology for modern biological and medical research. Various techniques have been developed for performing cellular assays. Single Cell Raman Spectroscopy (SCRS) is one of these. The method has the advantages of high resolution, no labeling, no damage, molecular specificity, no culture and the like. Single Cell Raman Spectroscopy (SCRS) is a highly efficient technique for identifying intracellular chemical substance information, providing information on the composition and structure of intracellular chemical molecules, and raman spectroscopy can detect the chemical substance fingerprint of a whole single cell without any markers and chemical bonds, thus identifying the cell type, physiological characteristics and phenotype changes of living cells, and tracking and sorting "unknown cell phenotypes" by using the changes of cell raman signals. But acquisition of single cell raman spectra can be disturbed by the raman background of the liquid. If amino acids, saccharides or the like are present in the environment of the cells to be measured, the measurement values of the cells to be measured are affected. Therefore, the cells to be detected need a certain pretreatment, and then the treated cells or bacteria and other particles are detected. The traditional cell pretreatment method mainly comprises two steps, namely centrifuging cells or bacteria, performing multi-step precipitation after centrifuging, removing supernatant, adding buffer solution for cleaning, and filtering the cells or bacteria on filter paper based on a filtering mode, and transferring, collecting and cleaning the cells or bacteria on the filter paper. And carrying out Raman detection after finishing the pretreatment. However, for complex samples, the treatment is more complicated, the activity and state of cells are affected by the traditional off-line cleaning means, the requirement of on-line detection, such as detection and monitoring of bacterial states in bacterial fermentation processes, is difficult to meet, and meanwhile, downstream analysis of inactivated cells is not performed any more. Disclosure of Invention In order to realize compatible analysis of various upstream complex samples and facilitate downstream cell analysis, the application provides a microfluidic chip, a preparation method and an online analysis device. The term "microparticle" as used herein refers to particles capable of being suspended in a non-organic phase solution (e.g., an aqueous phase) and passing within the microfluidic chip of the present invention, and includes particles of biological and non-biological origin, such as eukaryotic cells, prokaryotic cells, unicellular organisms, viral particles, organelles, particles formed of biological macromolecules, drug particles, drug carrier particles, liposomes, polymer particles, and the like. In a first aspect, the present application provides a microfluidic chip, which is implemented by adopting the following technical scheme. The micro-fluidic chip comprises a main channel, two or more sample injection channels are arranged in communication with the main channel, a focusing electrode and a deflection electrode are arranged at the bottom of the main channel, the focusing electrode is used for gathering particles, and