CN116144458A - Biological micro-fluidic chip and application thereof - Google Patents

Biological micro-fluidic chip and application thereof Download PDF

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Publication number
CN116144458A
CN116144458A CN202310135935.XA CN202310135935A CN116144458A CN 116144458 A CN116144458 A CN 116144458A CN 202310135935 A CN202310135935 A CN 202310135935A CN 116144458 A CN116144458 A CN 116144458A
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micro
channel
disc
bin
biological
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王�琦
罗维湘
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Changsha Pufangde Medical Equipment Co ltd
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Changsha Pufangde Medical Equipment Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/10—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by centrifugation ; Cyclones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

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Abstract

The invention relates to a micro-fluidic chip technology, in particular to a biological micro-fluidic chip and application thereof, wherein the biological micro-fluidic chip comprises a disc capable of centrifugally rotating, at least one group of biological sample processing units are arranged on the disc, each group of processing units comprises at least one containing bin and at least one collecting bin which are arranged on the disc, each containing bin is communicated with the corresponding collecting bin through a micro-channel arranged in the disc, biological samples in one containing bin are layered after being rotationally centrifuged by the disc, and one layer of liquid enters the corresponding collecting bin after being separated by the micro-channel. The invention arranges a plurality of containing bins and collecting bins on the disc of the microfluidic chip, and can communicate the collecting bins with the corresponding containing bins, thereby integrating the steps of separating, enriching, fluorescent staining, in situ hybridization, tabletting and the like of the circulating tumor cells on one disc in series, greatly saving the time of detecting and analyzing the circulating tumor cells and improving the efficiency.

Description

Biological micro-fluidic chip and application thereof
Technical Field
The invention relates to a micro-fluidic chip technology, in particular to a biological micro-fluidic chip and application thereof.
Background
The microfluidic chip technology (Microfluidics) integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a micron-scale chip, and automatically completes the whole analysis process. Because of its great potential in biological, chemical, medical and other fields, it has been developed into a new research field that has been crossed by the disciplines of biology, chemistry, medicine, fluids, electronics, materials, mechanics, etc. The microfluidic chip has the characteristics of controllable liquid flow, extremely small consumption of samples and reagents, ten times or hundreds times higher analysis speed and the like, can simultaneously analyze hundreds of samples in a few minutes or even shorter, and can realize the whole pretreatment and analysis processes of the samples on line. The ultimate goal of its application is to implement a micro total analysis system-Lab on a Chip (Lab on a Chip).
The most representative biological micro-fluidic chip is pregnancy diagnosis test paper, the detection reagent is prepared into HCG detection test paper by using an immunochromatography double-antibody sandwich method principle, and HCG in a urine sample can be detected within 3 minutes. During detection, when a urine sample to be detected siphons through the colloidal gold labeled antibody, an antigen-antibody colloidal gold complex is formed, the complex continues to crawl, a double-antibody sandwich colloidal gold complex is formed when passing through the coated anti-HCG alpha subunit Mca, a color band is presented at a coating line, excessive colloidal gold antibody continues to crawl, and a goat anti-mouse contrast linear Cheng Jiaoti gold immune complex is presented, and the color band is presented, so that whether a woman is pregnant or not can be diagnosed.
At present, circulating tumor cells (circulating tumor cells, CTCs) are separated and enriched from blood, and cells with different densities are separated by centrifugation, so that a centrifugal chip is a non-two option for realizing full-automatic enrichment and CTC identification. In a centrifugal CD microfluidic chip, the microchannels are distributed along the radial direction of the disc, which coincides with the direction of action of the centrifugal force (radially outwards). The fluid is pre-contained in a reservoir near the center of the disc, and when the disc is rotated by the motor, the fluid moves away from the center turning edge along the network of micro-channels under the action of centrifugal force. However, the existing CD microfluidic chip is only applied to the centrifugal separation step in the extraction process of the circulating tumor cells, so that the detection and analysis efficiency of the circulating tumor cells is low.
Disclosure of Invention
Aiming at the technical problems, the invention provides a biological micro-fluidic chip with higher detection and analysis efficiency and application thereof in the steps of separation, enrichment, fluorescent staining, in-situ hybridization and the like of circulating tumor cells.
The technical scheme adopted for solving the technical problems is as follows: the biological micro-fluidic chip comprises a disc capable of centrifugally rotating, wherein at least one group of biological sample processing units are arranged on the disc, each group of processing units comprises at least one containing bin and at least one collecting bin which are arranged on the disc, each containing bin is communicated with the corresponding collecting bin through a micro-channel arranged in the disc, biological samples in one containing bin are layered after being rotationally centrifuged through the disc, and one layer of liquid enters the corresponding collecting bin after being separated through the micro-channel.
Preferably, the micro-channel is a capillary micro-channel with a siphon effect, the first section of the micro-channel is communicated with the middle of the accommodating bin, the last section of the micro-channel is communicated with the top of the collecting bin, and the first section and the last section are communicated through an arc transitional middle section.
Preferably, each group of processing units further comprises at least two containing chambers and at least one collecting chamber, wherein one layer of liquid collected in the collecting chamber flows into the other containing chamber through the other micro-flow channel under the action of the rotating centrifugal force of the disc, and the liquid in the containing chamber and the substances in the other containing chambers are mixed and then enter the corresponding collecting chamber.
Preferably, the magnetic beads or the reagent are placed in the other accommodating chambers, and one layer of liquid collected in the collecting chambers and the magnetic beads or the reagent flow into the wavy micro-flow channels at the same time to be mixed and magnetically attracted or mixed and then enter the corresponding collecting chambers.
The invention also provides an application of the biological micro-fluidic chip in separating circulating tumor cells, wherein the biological sample is body fluid, the body fluid is layered after the disc is rotated and centrifuged, and a supernatant layer containing the circulating tumor cells automatically flows into a collecting bin after being separated by the micro-flow channel.
The invention further provides an application of the biological micro-fluidic chip in enriching the circulating tumor cells, wherein one collecting bin is an enriching bin, and after the supernatant containing the circulating tumor cells in one containing bin and the magnetic beads in the other containing bins flow into the micro-channel at the same time to be mixed and magnetically absorbed, the cell sediment containing the circulating tumor cells flows into the enriching bin.
Preferably, the micro flow channel is divided into two sections, one section communicated with at least two accommodating chambers is in a rotary shape, the other section communicated with the enrichment chamber is in a straight line shape, and at least one side of the straight line-shaped other section is provided with a magnet.
The invention further provides an application of the biological micro-fluidic chip in extracting circulating tumor cells, which comprises the steps of separation, enrichment, fluorescent staining, in-situ hybridization and tabletting, wherein after body fluid is separated and enriched, the fluorescent staining step is that cell sediment after magnetic attraction enrichment flowing into one of the accommodating chambers and fluorescent staining reagent in the other accommodating chambers flow into a micro-channel, fluorescent staining is mixed in the micro-channel, and the mixture flows into a corresponding collecting chamber after cleaning.
Preferably, the in situ hybridization step is to mix the washed cell sediment flowing into one of the accommodating chambers from the collecting chamber with the probe reagent flowing into the micro-flow channel in other accommodating chambers for in situ hybridization, and then wash and flow out.
Preferably, the disc is provided with at least two layers, the steps of separation, enrichment, fluorescent staining and in-situ hybridization are arranged on one or more layers, the step of tabletting is arranged on a bottom layer, the bottom layer is transparent and is provided with a cavity, the bottom layer is provided with a channel and a liquid injection hole which are communicated with the cavity, the cell sediment after in-situ hybridization cleaning flows into the cavity through the channel, and the chromogenic liquid injected through the liquid injection hole is mixed with the cell sediment in the cavity to finish tabletting.
According to the technical scheme, the plurality of containing bins and the collecting bin are arranged on the disc of the microfluidic chip, and the collecting bin is communicated with the corresponding containing bin, so that the steps of separating, enriching, fluorescent staining, in situ hybridization, tabletting and the like of circulating tumor cells can be integrated on one disc in series, and a plurality of samples can be processed simultaneously through the array processing unit, so that the time for detecting and analyzing the circulating tumor cells is greatly saved, and the efficiency is improved; the microfluidic chip is convenient to use and simple to operate, and the steps of enrichment, in-situ hybridization, fluorescent staining and the like are avoided after separation in the prior art.
Drawings
Fig. 1 is a schematic structural diagram of a preferred mode of the front surface of the microfluidic chip of the present invention.
Fig. 2 is a schematic view of a partial cross-sectional structure of a microfluidic chip according to the present invention.
Fig. 3 is a schematic diagram of an application flow of the microfluidic chip of the present invention.
Description of the embodiments
The present invention will now be described in detail with reference to the accompanying drawings, wherein the exemplary embodiments and descriptions of the present invention are provided for illustration of the present invention, but are not intended to be limiting.
As shown in fig. 1 and 2, the present invention provides a biological micro-fluidic chip, which comprises a centrifugally rotatable disk 1 mounted on a centrifuge for rotation therewith; at least one group of biological sample processing units 2 are arranged on the disc, and a plurality of groups of processing units can be arranged according to the area of the disc so as to process a plurality of biological samples at the same time, thereby improving the efficiency; each group of processing units comprises at least one containing bin 21 and at least one collecting bin 22 which are arranged on the disc, each containing bin is communicated with the corresponding collecting bin through a micro-channel 23 arranged in the disc, biological samples in one containing bin are layered after being rotated and centrifuged by the disc, and one layer of liquid enters the corresponding collecting bin after being separated by the micro-channel. According to the scheme, the designated liquid layer in the sample can be separated to the collection bin by utilizing the siphon action of the micro-channel, such as separating a supernatant layer containing circulating tumor cells in body fluid, so that the separation of biological samples is realized.
In the implementation process, the micro-channel adopts a capillary micro-channel with a siphon effect, the first section of the micro-channel is communicated with the middle of the accommodating bin, the last section of the micro-channel is communicated with the top of the collecting bin, and the first section and the last section are communicated through an arc-shaped transitional middle section. Specifically, the first section and the last section are all arranged in an outward inclined manner, the middle section is in an upward protruding arc shape, and the inclination angle, the arc shape radian, the length, the diameter and the like of each section are all obtained through calculation, so that the micro-channel has the function of a capillary tube, and the automatic separation of a liquid layer is realized under the siphon effect.
Preferably, each group of processing units comprises a collecting bin and at least two containing bins, wherein one containing bin is used for containing a sample to be processed, the other containing bins are used for containing magnetic beads or reagents, and the sample and the magnetic beads or the reagents flow into the micro-flow channel at the same time to be mixed and magnetically attracted or mixed and then enter the collecting bin. The disk of the present invention may be subjected to the mixing or mixing magnetic step alone. When one of the accommodating chambers is used for accommodating a sample to be processed and the other accommodating chambers are used for accommodating reagents, the sample and the reagents enter the micro-channel simultaneously under the action of centrifugal force, are fully mixed in the wavy rotary micro-channel and flow into the collecting chamber, and the steps such as fluorescent staining, in-situ hybridization and the like in the process of extracting circulating tumor cells can be independently completed; when one of the accommodating chambers is used for accommodating a sample to be processed and the other accommodating chambers are used for accommodating magnetic beads, the sample and the magnetic beads enter the micro-channel simultaneously under the action of centrifugal force, after being mixed in the wavy rotary micro-channel, the magnetic beads are adsorbed by the magnet 24 arranged on the disc, so that specified components in the sample flow into the collecting chamber, and the mixed magnetic attraction is realized, for example, the enrichment step in extracting circulating tumor cells can be independently completed.
Each group of processing units further comprises at least two containing bins and at least one collecting bin, wherein one layer of liquid collected in the collecting bin flows into one containing bin through the micro flow channel under the action of the rotating centrifugal force of the disc, and the liquid in the containing bin and the magnetic beads or the reagent in the other containing bins flow into the micro flow channel to be mixed and magnetically attracted or mixed and then enter the corresponding collecting bin. That is, after the above-mentioned separation step is performed on the disk, a mixing magnetic attraction or mixing step may be continuously performed on the disk. For example, after the biological separation of the circulating tumor cells, magnetic enrichment, fluorescent staining, in situ hybridization and the like are continuously carried out; other non-magnetic attraction steps may also be performed directly in succession after separation.
The invention provides an application of a biological micro-fluidic chip in separating circulating tumor cells, which comprises the following steps: the containing bin is used for containing body fluid, the body fluid to be collected is added into the upper layer of the isolating liquid, the body fluid is layered after the disc rotates and is centrifuged, and the supernatant layer containing circulating tumor cells automatically flows into the collecting bin after being separated by the capillary micro-flow channel with siphoning effect, so that the separation of the circulating tumor cells is realized. Wherein the body fluid comprises at least one of pleural effusion, peritoneal effusion, cerebrospinal fluid, lymph fluid, blood of a human or animal.
The invention provides an application of a biological micro-fluidic chip in enriching circulating tumor cells, which is characterized in that one collecting bin is an enriching bin, and after supernatant containing circulating tumor cells in one containing bin and magnetic beads in the other containing bins flow into a micro-channel to be mixed and magnetically absorbed, cell sediment containing the circulating tumor cells flows into the enriching bin. In the implementation process, the supernatant in one containing bin can be from the supernatant obtained by adopting other independent separation technologies, or can be from the supernatant layer in the collecting bin in the separation step of the disc, so that the continuous separation and enrichment steps of circulating tumor cells are realized. Preferably, the micro-flow channel is divided into two sections, and one section communicated with at least two accommodating chambers is in a rotary shape, so that repeated mixing of magnetic beads and supernatant is facilitated; the other section communicated with the enrichment bin is in a straight line shape, and at least one side of the straight line-shaped other section is provided with a magnet 24, so that magnetic beads in the micro-channel and white blood cells on the magnetic beads can be adsorbed, and cell sediment containing tumor cells is obtained.
As shown in FIG. 3, the invention provides an application of a biological micro-fluidic chip in extracting circulating tumor cells, which comprises the steps of separation, enrichment, fluorescent staining, in-situ hybridization and tabletting on a disc, specifically, after body fluid is separated and enriched, the fluorescent staining step is that cell sediment after magnetic enrichment flowing into one of the accommodating chambers and fluorescent staining reagent flowing into the other accommodating chamber flow into a micro-channel, fluorescent staining is mixed in the micro-channel, and then the mixture flows into a corresponding collecting chamber after cleaning. The washing step is also carried out on the disc, namely, the cell sediment after fluorescent staining flows into the accommodating bin, the other accommodating bin is used for accommodating the washing reagent, and the cell sediment and the cleaning reagent are mixed in the micro-channel, washed and layered, then enter the collecting bin, and then in-situ hybridization is carried out. The in situ hybridization step is to mix the washed cell sediment flowing into one of the accommodating chambers from the collecting chamber with the probe reagent flowing into the micro flow channel from the other accommodating chambers for in situ hybridization, and then wash the mixture for two times and flow out. The cleaning steps are the same as above. Thereby realizing continuous in situ hybridization and fluorescent staining steps.
Preferably, the disc is provided with at least two layers, and the steps of separation, enrichment, fluorescent staining and in situ hybridization are arranged in one or more layers, namely the four steps of separation, enrichment, fluorescent staining and in situ hybridization can be distributed along the disc surface of the disc, and only one layer is needed; one or two of the four steps can be arranged on one layer respectively, namely four, three, two layers and the like are adopted; the step of tabletting is arranged on a bottom layer 25, the bottom layer is transparent and is provided with a cavity 26, the bottom layer is provided with a channel 27 and a liquid injection hole 28 which are all communicated with the cavity, the cell sediment after in situ hybridization cleaning flows into the cavity through the channel, and the chromogenic liquid injected through the liquid injection hole and the cell sediment are mixed in the cavity to finish tabletting. The bottom layer is made of transparent materials, and the mixture in the cavity is directly scanned by a scanner below the bottom layer to detect and analyze the number of the circulating tumor cells, so that the full-flow detection and analysis of the circulating tumor cells are realized on the disc. The invention integrates the steps of separating, enriching, fluorescent staining, in situ hybridization, tabletting and the like of the circulating tumor cells on one disc in series, thereby greatly improving the detection and analysis efficiency.

Claims (10)

1. A biological micro-fluidic chip, includes the disc that can centrifugal rotation, its characterized in that: the biological sample collection device comprises a disc, and is characterized in that at least one group of biological sample processing units are arranged on the disc, each group of processing units comprises at least one containing bin and at least one collecting bin which are arranged on the disc, each containing bin is communicated with the corresponding collecting bin through a micro-channel arranged in the disc, biological samples in one containing bin are layered after rotating and centrifuging the disc, and one layer of liquid enters the corresponding collecting bin after being separated through the micro-channel.
2. The biological microfluidic chip according to claim 1, wherein: the micro-channel is a capillary micro-channel with a siphon effect, the first section of the micro-channel is communicated with the middle of the accommodating bin, the last section of the micro-channel is communicated with the top of the collecting bin, and the first section and the last section are communicated through an arc-shaped transitional middle section.
3. The biological microfluidic chip according to claim 1 or 2, characterized in that: each group of processing units further comprises at least two containing bins and at least one collecting bin, wherein one layer of liquid collected in the collecting bin flows into the other containing bin through the other micro-channels under the action of the rotating centrifugal force of the disc, and the liquid in the containing bin is mixed with substances in the other containing bins and then enters the corresponding collecting bin.
4. A biological micro fluidic chip according to claim 3, characterized in that: the magnetic beads or the reagents are placed in the other accommodating chambers, and one layer of liquid collected in the collecting chambers and the magnetic beads or the reagents flow into the wavy micro-flow channels to be mixed and magnetically attracted or mixed and then enter the corresponding collecting chambers.
5. Use of the biological micro-fluidic chip according to any one of claims 1 to 4 for separating circulating tumor cells, characterized in that: the biological sample is body fluid, the body fluid is layered after the disc is rotated and centrifuged, wherein a supernatant layer containing circulating tumor cells is separated by the micro-channel and automatically flows into the collecting bin.
6. Use of the biological micro-fluidic chip according to claim 3 or 4 for enriching circulating tumor cells, characterized in that: one of the collecting chambers is an enriching chamber, and the supernatant containing the circulating tumor cells in one of the containing chambers and the magnetic beads in the other containing chambers flow into the micro-channel to be mixed and magnetically attracted, and then the cell sediment containing the circulating tumor cells flows into the enriching chamber.
7. The use of the biological micro-fluidic chip according to claim 6 for enriching circulating tumor cells, wherein the biological micro-fluidic chip comprises: the micro-flow channel is divided into two sections, one section communicated with at least two accommodating chambers is in a rotary shape, the other section communicated with the enrichment chamber is in a straight line shape, and at least one side of the straight line-shaped other section is provided with a magnet.
8. Use of the biological micro-fluidic chip according to any one of claims 1 to 4 for extracting circulating tumor cells, comprising the steps of separation, enrichment, fluorescent staining, in situ hybridization and tabletting, characterized in that: after body fluid is separated and enriched, the cell sediment after magnetic enrichment flowing into one of the accommodating chambers and the fluorescent staining reagent flowing into the other accommodating chambers flow into a micro-channel, fluorescent staining is mixed in the micro-channel, and the cell sediment flows into the corresponding collecting chamber after cleaning.
9. The use of the biological micro-fluidic chip according to claim 8 for extracting circulating tumor cells, wherein: the in situ hybridization step is to mix the cleaned cell sediment flowing into one of the accommodating chambers from the collecting chamber with the probe reagent flowing into the micro flow channel from the other accommodating chambers for in situ hybridization, and then clean and flow out.
10. The use of the biological micro-fluidic chip according to claim 9 for extracting circulating tumor cells, wherein: the disc is provided with at least two layers, the steps of separation, enrichment, fluorescent dyeing and in-situ hybridization are arranged on one layer or a plurality of layers, the step of tabletting is arranged on the bottom layer, the bottom layer is transparent and is provided with a cavity, the bottom layer is provided with a channel and a liquid injection hole which are communicated with the cavity, the cell sediment after in-situ hybridization cleaning flows into the cavity through the channel, and the chromogenic liquid injected through the liquid injection hole is mixed with the cell sediment in the cavity to finish tabletting.
CN202310135935.XA 2023-02-01 2023-02-01 Biological micro-fluidic chip and application thereof Pending CN116144458A (en)

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CN202310135935.XA CN116144458A (en) 2023-02-01 2023-02-01 Biological micro-fluidic chip and application thereof

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CN202310135935.XA CN116144458A (en) 2023-02-01 2023-02-01 Biological micro-fluidic chip and application thereof

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