CN210314199U - Micro-fluidic chip for rapidly capturing or detecting cells - Google Patents
Micro-fluidic chip for rapidly capturing or detecting cells Download PDFInfo
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- CN210314199U CN210314199U CN201920852315.7U CN201920852315U CN210314199U CN 210314199 U CN210314199 U CN 210314199U CN 201920852315 U CN201920852315 U CN 201920852315U CN 210314199 U CN210314199 U CN 210314199U
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Abstract
The utility model relates to a biological detection technical field discloses a micro-fluidic chip for catch fast or detect cell, and its structure includes at least a set of micro-fluidic unit, and the structure of micro-fluidic unit includes interconnect's passageway A and passageway B, is equipped with multiunit fishbone shape constitutional unit in the passageway A. The utility model discloses combine fishbone-shaped structure and cylinder microcavity structure, improved the high efficiency of target cell such as food-borne pathogenic bacteria or tumor circulating cell, low-cost, specific capture, enrichment and external multiplication efficiency, during the microcavity structure of very little volume is gathered to the cell that will catch, further improved the survival rate of external culture.
Description
Technical Field
The utility model relates to a biological detection equipment specifically is the micro-fluidic chip of catching or detecting cell fast.
Background
Food-borne diseases are generally diseases caused by ingestion of biological, chemical, or physical harmful substances that enter the human body with food or drinking water. In recent years, the proportion of food-borne diseases caused by microorganisms in food safety events is continuously increased, which poses serious threats to human health and life safety, enteropathogenic escherichia coli is a common pathogenic bacterium causing human diarrhea symptoms, and currently, the pathogenic escherichia coli is divided into 6 types according to different biological characteristics, namely enteropathogenic escherichia coli, enterotoxigenic escherichia coli, enteroinvasive escherichia coli, enterohemorrhagic escherichia coli, enteroaggregative escherichia coli, and enteroshiga-like toxin-producing and invasive escherichia coli discovered recently. Among them, enteropathogenic escherichia coli is an important pathogenic bacterium causing acute and chronic diarrhea in infants and diarrhea in adults all over the world. Pollution caused by staphylococcus aureus, salmonella and the like also seriously threatens the health of human beings, and acute gastroenteritis, vomiting, nausea, abdominal pain, diarrhea, fever and the like are caused by acute poisoning; serious patients can cause respiratory, circulatory and nervous system symptoms, and life can be threatened if rescue is not timely performed; chronic poisoning can lead to carcinogenesis, teratogenesis, mutagenesis, etc. Conventional bacteria detection methods, such as culture and isolation of microorganisms, have low sensitivity, long treatment time, and need trained personnel, and not all bacteria can be cultured in a laboratory, which has great limitations. Under the continuous development of scientific technology, new detection methods such as an analytical immunology method, a molecular biology detection technology, a magnetic fluorescence nano detection method and the like are generated, and the defects of the traditional detection method are overcome.
(1) The immunological analysis method is based on the specific reaction of antigen and antibody, and during the detection, the organism may be stimulated to produce specific antibody based on the specific antigen characteristic of microbe in food. The immunomagnetic bead separation method, the colloidal gold labeling method and the enzyme-linked immunosorbent assay (ELISA) all belong to the common detection methods of immunology. The immunomagnetic bead separation method is suitable for bacteria enrichment, and needs to be combined with other detection methods for bacteria detection; the colloidal gold labeling method has the characteristics of high sensitivity, strong specificity, simple operation and the like, but when the colloidal gold labeling method is applied to the detection of food-borne pathogenic bacteria microorganisms, false negative reaction is possibly generated because the antigen is masked by certain substances in a sample, and the accuracy and the authenticity of a detection result are difficult to ensure; the ELISA detection method has high sensitivity and strong specificity. The detection speed is high, but the operation is complex.
(2) The molecular biology detection technology is used for further researching molecules of microorganisms, particularly nucleic acid structures and related components thereof, from the molecular biology level, and comprises gene chip detection, PCR detection methods and the like. In the process of detecting the food-borne pathogenic microorganisms, the gene chip detection method improves the detection quality and efficiency by the characteristics of small reaction volume, small reagent consumption, high detection speed, high sensitivity and the like, but the detection method has high detection cost and low detection precision; the PCR detection method realizes qualitative to quantitative leap, and the sensitivity is high without subsequent treatment in the whole detection process. The detection result has high precision, but when the kit is applied to the detection of food-borne pathogenic bacteria microorganisms, the activity of enzyme in PCR (polymerase chain reaction) can be inhibited by the components in the food, so that the detection accuracy is reduced to a certain extent.
(3) The magnetic fluorescence nano detection method combines magnetic nano particles and fluorescent nano particles in a preset mode, so that the magnetic fluorescence nano particles and the fluorescent nano particles have double functions of marking and enriching, generate stronger fluorescence under the interaction, and provide better magnetic responsiveness, light resistance, optical signals and the like.
Malignant tumor is one of the most serious diseases in the world at present, and with the increasing incidence of tumor, people continuously deepen understanding and research on the malignant tumor, and more than 90 percent of the deaths of tumor patients are caused by tumor metastasis. Tumor cells, which are Circulating Tumor Cells (CTCs) that survive in the blood circulation system, fall off from the primary tumor site spontaneously or as a result of diagnostic procedures and enter the blood to form circulating tumor cells, can characterize the molecular characteristics of tumor lesions, and are considered as the main pathway for the metastasis of tumor blood. The detection of circulating tumor cells in the blood circulation system implies cancer and possible metastasis of the cancer. Therefore, the capture of the CTCs with early, high efficiency and specificity is not only a powerful means for early diagnosis of tumors, but also facilitates risk assessment and personalized treatment scheme before tumor metastasis occurs by releasing the obtained CTCs and carrying out deep research and analysis on the biological activity, molecules and functions of the CTCs.
However, the current researches on capturing and activity analysis of circulating tumor cells face some problems: 1. the number of CTCs in peripheral blood of cancer patients is very rare, with as many as 1 million white blood cells and 50 million red blood cells per milliliter of blood, but only a few to hundreds of CTCs, and studies have shown that these CTCs have great heterogeneity, particularly functional heterogeneity; 2. the method has the advantages that irreversible damage is caused to CTCs in the pretreatment and enrichment processes of blood samples, apoptosis is caused, and the biological activity research in the later period is influenced, so that the real-time treatment strategy evaluation aiming at patients is influenced. Therefore, how to efficiently isolate CTCs from peripheral blood of cancer patients becomes one of the major challenges facing tumor "liquid biopsy".
Many enrichment techniques have been developed to capture and isolate CTCs, each of which is based on certain characteristics to isolate and enrich CTCs. It is mainly divided into two major methods based on the physical and biological properties of tumor cells, and the most common separation method in the latter is immunomagnetic separation technology. The magnetic particles have high specificity and enrichment separability under the action of a magnetic field, and are widely used for detecting CTCs. The separation principle is that after the magnetic beads are combined with specific antibodies, the magnetic beads are identified with cells containing specific antigens to form cell antigen-antibody-magnetic bead immune complexes. The compound moves under the action of magnetic force, and the separation of CTCs from other substances can be realized. The CellSearch system kit of the Johnson corporation in America based on the principle is the only system which is approved by the Food and Drug Administration (FDA) of America and applied to the detection of CTCs at present. The kit has the advantages of high sensitivity and specificity, but has the defects of high price, incapability of being popularized and used in a large amount clinically and limited capture efficiency; in addition, magnetic particle-based CTCs separation capture also exhibits some disadvantages in several respects: 1. depending on the tumor associated antigen with high specificity, part of the CTCs can be lost in the capturing process; 2. tumor cells are easy to aggregate in a magnetic field, mechanical damage is caused to the cells, and the cell morphology and activity are influenced; 3. cells bound to magnetic beads are difficult to separate and may interfere with subsequent detection.
Therefore, the microfluidic technology is a technology for manipulating or controlling a liquid in a micro-scale flow channel, and has the advantages of low reagent sample consumption, rapidness, low cost, high throughput, simplicity and convenience in use, and the like.
Microfluidic technology is a promising tool for the capture or detection of bacterial cells. With the increasing maturity of DNA nanotechnology, aptamers have been developed for detection of pathogenic bacteria in recent years because of their small size, stability, low production cost, and easy modification of specific groups at their ends during chemical synthesis, and the modified end groups help to effectively fix these molecules on different sensors.
The micro-fluidic chip is used for capturing circulating tumor cells, and capturing methods can be divided into a physical method and a biochemical method. In the current common and classical applications, the methods belong to physical methods such as micro-structure filtration, inertial sorting, deterministic lateral displacement and the like, and biochemical methods such as an immunomagnetic bead capture method by magnetic field force and a passive capture method without external force field. The fishbone-shaped microfluidic chip can break a laminar flow state at a low Reynolds number, so that a streamline is in a spiral state to achieve the aim of liquid mixing, and is used for DNA molecular hybridization, antigen-antibody binder particle sorting and the like; there are also studies on their use for capturing CTCs. Nevertheless, there are still some technical obstacles in the current sorting technology of CTCs based on microfluidic technology: 1. although the microfluidic chip immobilized with the capture antibody has better CTCs capture capacity, the release of CTCs for subsequent analysis is more difficult; 2. when cell sorting is carried out, certain damage can be caused to the biological activity of cells due to shearing force in a channel or collision with a tube wall; 3. the technology is mostly used for sorting and enriching the CTCs at present, but the research of in vitro culture of the CTCs by utilizing the microfluidic chip technology is not complete, and the integrated research of collection, release and culture on the chip is less.
Therefore, the defects of the prior art need to be overcome, the specific capture, detection, release and enrichment culture of CTCs in a whole blood sample or microorganisms, especially food-borne pathogenic bacteria, in a food sample is realized through the design of a microfluidic chip channel, the damage of bacteria or circulating tumor cells in the capture process is reduced, and the rapid enrichment, detection and in-vitro culture of target cells are realized.
SUMMERY OF THE UTILITY MODEL
The utility model aims at providing a micro-fluidic chip for catch or detect cell fast.
A microfluidic chip for rapidly capturing or detecting cells structurally comprises at least one group of microfluidic units, wherein the number of the microfluidic units is 4-8, and the microfluidic units are arranged in an annular array. Preferably, the number of the microfluidic units is 6, and the microfluidic units are arranged in an annular array.
Each group of micro-fluidic units structurally comprises a channel A and a channel B which are mutually connected, wherein the channel A and the channel B are snake-shaped channels;
one end of the channel A is provided with a sample inlet I, and the sample inlet I is positioned on the outer side of the microfluidic chip; and a sample inlet II is arranged at the joint of the channel A and the channel B, and the tail end of the channel B is a sample outlet which is positioned at the inner side of the microfluidic chip.
The channel A and the channel B are snakelike and comprise a linear channel and an arc-shaped channel. The straight-line channels of the channel A and the channel B are arranged in parallel, and the adjacent straight-line channels are connected through an arc-shaped channel; in each group of micro-fluidic units, the length of each linear channel is different, and the length of each linear channel gradually increases from the center of the micro-fluidic chip to the outside. The straight channel lengths of the channels B are all smaller than the straight channel length of the channel A.
And the sample inlet II is positioned on the arc-shaped channel connecting the channel A and the channel B.
The depth of the channel A and the channel B is 80-200 mu m, and the width of the channel A and the channel B is 0.8-1.5 mm.
A plurality of groups of fishbone-shaped structural units are arranged in the passage A; the fishbone-shaped structural unit is positioned at the top in the linear channel of the channel A.
Each group of fishbone-shaped structural units comprises a plurality of fishbones which are parallel to each other, and fishbone gaps are formed between every two adjacent fishbones. Each group of fishbone-shaped structural units comprises 6-12 fishbones, preferably 8-12 fishbones. The width of the fishbone is 30-50 mu m; taking the top of the channel A as a reference, and sinking the fishbone upwards to a depth of 30-50 mu m; the depth of the fishbone is the height of the fishbone gap; in the fishbone-shaped structural units of the same group, the distance between adjacent fishbones (i.e. the width of fishbone gaps) is 80-120 μm.
The fishbone structure comprises a fishbone micro groove A and a fishbone micro groove B, the included angles between the fishbone micro groove A and the fishbone micro groove B and the straight line channel of the channel A are respectively 30-60 degrees, and the included angle between the fishbone micro groove A and the fishbone micro groove B is 80-100 degrees; the length of the fishbone microgrooves A is not equal to that of the fishbone microgrooves B. The direction of all the fishbones (i.e. the direction of the included angle formed by the fishbone microgrooves A and B) is the same as or opposite to the flowing direction of the fluid, taking the flowing direction of the fluid as the positive direction.
The lengths of the fishbone microgrooves A and the fishbone microgrooves B of the fishbone-shaped structural units are distributed at intervals according to the proportion of 1: 1.8-2.1 and 1.8-2.1: 1 on the same straight-line channel in the advancing direction of the fluid.
On the same straight line channel, the shortest distance between adjacent fishbone-shaped structural units is 80-120 mu m. Namely, the shortest distance between the fishbone microgrooves A or B of the adjacent fishbone-shaped structural units is 80-120 μm.
And a micro-cavity structure is arranged in the channel B and is positioned at two sides of the inner wall of the linear channel of the channel B. The microcavity structures are cylindrical microcavity structures, the cylindrical microcavity structures are distributed in an array mode at adjacent intervals of 80-120 mu m, and the number of the cylindrical microcavity structures on each linear channel changes along with the length of the linear channel of the channel B. The central shaft of the cylindrical micro-cavity structure is vertical to the microfluidic chip and the bottom of the linear channel of the channel B, and is vertical to the fluid direction of the linear channel of the channel B; each cylindrical microcavity structure is connected to the channel B straight channel through a small rectangular channel at the bottom.
Preferably, the diameter of the cylindrical micro-cavity structure of the channel B is 80-200 μm, and the depth is 80-200 μm. The length of the rectangular small channel is 30-40 m, and the depth of the rectangular small channel is 30-40 mu m.
In a preferred embodiment of the present invention, the number of the microfluidic channel units is 6, and the microfluidic channel units are arranged in a circular array. The depth of channels A and B was 100 μm and the width 1 mm. The diameters of the sample inlet I, the sample inlet II and the sample outlet are 1 mm; the depth of the fishbone is 35 μm and the width is 35 μm by taking the top of the channel A as a reference; in the fishbone-shaped structural units of the same group, the distance between adjacent fishbones is 100 mu m; on the same straight line channel, the shortest distance between the adjacent fishbone-shaped structural units is 100 mu m; each group of fishbone-shaped structural units comprises 10 fishbones, the included angles between the fishbone microgrooves A and B of the fishbones are 90 degrees, and the included angles between the fishbone microgrooves A and the fishbone microgrooves B and the two sides of the channel A are 45 degrees respectively; the lengths of the fishbone microgrooves A and the fishbone microgrooves B of the fishbone-shaped structural units are distributed at intervals in the ratio of 1:2 and 2:1 on the same straight line channel or in the advancing direction of the fluid. The diameter of the cylindrical microcavity structure in the channel B is 100 micrometers, the depth of the cylindrical microcavity structure in the channel B is 100 micrometers, and the distance between adjacent cylindrical microcavity structures is 100 micrometers; the rectangular mini-channels have a depth of 35 μm and a width of 35 μm.
When the method is used, according to the conventional operation, firstly, sulfydryl is modified on the inner wall of the channel A, then one vertex of a DNA tetrahedron is modified by biotin, and the other one to three vertices of the DNA tetrahedron are modified by amide, and the DNA tetrahedron is fixed on the inner surface of the channel A through the Michael addition reaction. Fixing a specific aptamer sequence for identifying/capturing target cells on the top of a DNA tetrahedron modified with biotin through avidin, connecting a biotin-modified specific aptamer capable of capturing the target cells through avidin, and realizing the construction of a DNA tetrahedron-avidin-aptamer structure, wherein the structure contains enzyme cutting sites, so that the sequence of the specific aptamer is connected to the inner surface of a channel A.
During detection, a sample is added from a sample inlet I, negative pressure is applied to a sample inlet II to realize sample introduction, waste liquid is sucked out, and target cells are captured by an aptamer on a channel A;
after the target cell is captured, adding DNase into the channel A from the sample inlet I under the negative pressure condition, incubating and carrying out enzyme digestion, and cutting the channel A from the enzyme digestion site of the modification sequence to release the captured target cell;
and after the sample outlet is subjected to negative pressure treatment, the sample inlet II is sealed and the sample outlet is opened, and the target cells are released and then flow into the cylindrical microcavity structures on two sides in the channel B between the sample inlet II and the sample outlet along with the enzyme digestion liquid for enrichment.
Detecting cells in the cylindrical microcavity structure can identify whether the sample contains target cells.
Alternatively, cell culture medium is added into the channel B for incubation, and cells in the cylindrical microcavity structure are observed to determine whether the sample contains the target cells.
The utility model discloses a micro-fluidic chip can realize target cell, for example tumor cell or microbial cell's specific capture, detection, release and enrichment culture, reduces target cell and in the damage of catching the in-process, improves the sensitivity and the specificity that detect, realizes short-term test.
Although rapid cell detection techniques have been developed rapidly in recent years, there are still significant limitations to achieving rapid, sensitive, and low-cost detection. Therefore, the utility model provides a micro-fluidic chip for cell rapid detection, the fishbone structure in the chip can break the laminar flow state when the fluid flows in the serpentine channel and the Reynolds number is low, and vertical flow is generated, so that the streamline presents a spiral form, thereby being beneficial to the mixing and uniform mixing of the fluid in the channel; the part from a sample inlet I to a sample inlet II of a snake-shaped channel of the microfluidic chip is modified by a DNA tetrahedron-avidin-aptamer; the existence of the fishbone-shaped structure in the chip increases the collision probability between the cell and the wall surface modified by the DNA tetrahedron-avidin-aptamer structure, reduces the shearing force borne by the cell, better maintains the cell activity of the captured target cell, increases the cell capture rate, and reduces the irreversible damage to the target cell in the capture process.
After the target cell is captured, DNA enzyme is added into the chip channel at the injection port I, the action temperature and time are controlled, the aptamer sequence is cut at the enzyme digestion site, the injection port 2 is sealed, negative pressure treatment is carried out at the sample outlet, the captured cell is released and flows into the microcavity structures at two sides of the channel between the injection port II and the sample outlet along with enzyme digestion liquid, the enrichment of the cell is realized, and the detection and in-vitro culture can be carried out.
The utility model discloses a micro-fluidic chip combines fishbone-shaped structure and microcavity structure, can with the cell from catching culture systematization, the high efficiency of target cell such as food-borne pathogenic bacteria or tumour circulating cell has been realized, low cost, high sensitivity, high specificity is caught, detect, release and enrichment, combine fishbone-shaped structure and cylinder microcavity structure, the capture and the external proliferation efficiency of cell have been improved, enrich the microcavity structure of very little volume with the cell of catching, the survival rate of in vitro culture has further been improved, the effectual external amplification of target cell in the sample has been realized, thereby molecular analysis and the functional study for further realizing the cell have laid a foundation.
Drawings
FIG. 1 is a design drawing of a microfluidic chip
FIG. 2 is a design drawing of a microfluidic channel unit
FIG. 3 is a schematic view of a serpentine channel without fishbone-shaped structural units and cylindrical microcavity structures
FIG. 4 is a top view of the fishbone-shaped structural unit of channel A
FIG. 5 is a longitudinal section of a channel A with a fishbone-shaped structural unit
FIG. 6 is a top view of the cylindrical microcavity structure of channel B
FIG. 7 is a view of the microcavity structure of the cylinder of channel B in the direction of FIGS. 6C-C
FIG. 8 shows a schematic diagram (A) of a microcavity structure of a cylinder of channel B and a schematic diagram (B) of a fishbone-shaped structural unit in channel A in example 1
FIG. 9 is a graph showing fluorescence detection of cells in a single cylindrical microcavity structure of example 3
FIG. 10 is the results of bright field and fluorescence measurements in the microcavity structure of a single cylinder after the addition of different cell suspensions for detection in example 3
FIG. 11 is the result of 0 to 18 hours of culture of the enriched cells in example 3
1-channel A, 101-fishbone-shaped structural unit, 102-fishbone, 103-fishbone gap, 104-fishbone microgroove A, 105-fishbone microgroove B, 107-channel A linear channel, 108-channel A arc channel, 2-channel B, 201-cylinder microcavity structure, 202-rectangular small channel, 207-channel B linear channel, 208-channel B arc channel, 3-microflow channel unit, 4-sample inlet I, 5-sample inlet II, 6-sample outlet.
Detailed Description
Example 1
As shown in fig. 1, 6 groups of microfluidic units are distributed on the microfluidic chip and arranged in a ring array.
The design of the microfluidic unit is shown in fig. 2, and the structure comprises a channel A and a channel B which are connected with each other, wherein the channel A and the channel B are serpentine channels, and the cross sections of the channels are rectangular; the serpentine channel without the fishbone-shaped structural elements and the cylindrical microcavity structure is shown in FIG. 3.
The channel A and the channel B are both snakelike and comprise a linear channel and an arc-shaped channel; and the straight channels of the two are arranged in parallel; the length of each linear channel is unequal, and the length of each linear channel gradually increases from the center of the microfluidic chip to the outside; the straight channel lengths of the channels B are all smaller than the straight channel length of the channel A. The linear channels of the channel A are parallel to each other, and the adjacent linear channels 107 of the channel A are connected by an arc-shaped channel 108 of the channel A; the straight channels of the channel B are parallel to each other, and the adjacent straight channels 207 of the channel B are connected by an arc channel 208 of the channel B.
One end of the channel A is provided with a sample inlet I, and the sample inlet I is positioned on the outer side of the microfluidic chip; and a sample inlet II is arranged at the joint of the channel A and the channel B, and the tail end of the channel B is a sample outlet which is positioned at the inner side of the microfluidic chip. And the sample inlet II is positioned on the arc-shaped channel connecting the channel A and the channel B.
The depth of channels A and B was 100 μm and the width 1 mm. The diameters of the sample inlet I, the sample inlet II and the sample outlet are 1 mm.
As shown in fig. 3 and 4, a plurality of groups of fishbone-shaped structural units are arranged in the channel A; the fishbone-shaped structural unit is positioned at the top of the linear channel of the channel A. Each group of fishbone-shaped structural units comprises 10 fishbones which are parallel to each other, and fishbone gaps are formed between every two adjacent fishbones.
The structure of the fishbone comprises a fishbone micro-groove A and a fishbone micro-groove B, the included angles between the fishbone micro-groove A and the fishbone micro-groove B and the linear channel 107 of the channel A are 45 degrees respectively, and the included angle between the fishbone micro-groove A and the fishbone micro-groove B is 90 degrees; the length of the fishbone microgrooves A is not equal to that of the fishbone microgrooves B, and the length ratio is 1:2 or 2: 1. The lengths of the fishbone microgrooves A and the fishbone microgrooves B on the same straight line channel or in the advancing direction of the fluid are distributed at intervals in the proportion of 1:2 and 2: 1.
The direction of the fish bone is the same as the direction of fluid flow, as shown in fig. 4.
The width of the fishbone is 35 μm; taking the top of the channel A as a reference, the fishbone is sunken upwards, the depth is 35 mu m, and the distance from the top of the fishbone to the bottom of the channel A is 135 mu m; the distance from the lower part of the fish bone gap to the bottom of the channel a was 100 μm. As shown in fig. 5 and 8B.
In the same group of fishbone-shaped structural units, the distance between adjacent fishbones, namely the width of a fishbone gap, is 100 mu m, and on the same straight line channel, the shortest distance between the adjacent fishbone-shaped structural units is 100 mu m, namely the shortest distance between fishbone microgrooves A or fishbone microgrooves B of the adjacent fishbone-shaped structural units is 100 mu m. As shown in fig. 8B.
As shown in fig. 6, 7 and 8A, cylindrical microcavity structures are arranged in the channels B, the cylindrical microcavity structures with a diameter of 100 μm are sequentially distributed in an array at an adjacent interval of 100 μm on the inner walls of the two sides of each linear channel B, and the number of the cylindrical microcavity structures on each linear channel changes with the length of the linear channel B; the central shaft of the cylindrical micro-cavity structure is vertical to the microfluidic chip and the bottom of the linear channel of the channel B, and is vertical to the fluid direction of the linear channel of the channel B; each cylindrical microcavity structure communicates with the channel B straight channel through its bottom rectangular small channel 202. The rectangular small channel has a width of 35 μm, a length of 60 μm and a depth of 35 μm.
Example 2
In the embodiment 1, 3-methoxy-mercaptopropyl silane is firstly used for surface mercapto modification in a channel A between a sample inlet I and a sample inlet II of each microfluidic unit of the microfluidic chip to construct a DNA tetrahedron structure with the total length of 100bp, acrylamide is modified on three vertexes of the DNA tetrahedron structure, and the DNA tetrahedron is fixed on the inner surface of the channel through Michael addition reaction between the acrylamide and the mercapto group in the channel A. The other vertex of the DNA tetrahedron is modified with biotin.
Selecting an aptamer sequence capable of specifically recognizing Escherichia coli O157: H7 cells, wherein the aptamer sequence contains an EcoRI enzyme cutting site; and the construction of a DNA tetrahedral-avidin-aptamer structure is realized through avidin-biotin, and an aptamer sequence capable of capturing Escherichia coli O157: H7 cells is connected and fixed on the inner surface of the channel A.
Coli O157H 7 cells were stained with fluorescent light, resuspended in 1mL of phosphate buffer (pH 7.4) and prepared into 10 cells7、106、105、104、103、102CFU/mL of the mixture.
And opening the sample inlet II and introducing 100 mu L of cell suspension containing Escherichia coli O157: H7 cells into the microfluidic chip slowly from the sample inlet I under the negative pressure condition of-0.06 Mpa of vacuum degree to realize sample injection and waste liquid suction, wherein the target cells are identified and captured by the aptamer sequence in the channel A.
After the target cell is captured, adding an enzyme digestion buffer solution of EcoRI into the channel A from the sample inlet I, incubating for 1h at 37 ℃, cutting off the aptamer sequence at an enzyme digestion site, and releasing the captured cell from the channel A; sealing the sample inlet II, and applying negative pressure of-0.06 Mpa vacuum degree to the sample outlet for about 30 s; the enzyme digestion solution flows into the channel B under the negative pressure condition of-0.06 Mpa vacuum degree, and the escherichia coli flows into the cylinder microcavity structures at the two sides of the channel B along with the enzyme digestion solution, so that the escherichia coli is enriched and can be used for detection.
The fluorescence detection results are shown in FIG. 9. The enrichment effect can reach more than 70 percent according to the statistics of the number of cells in the sample injection.
The content of the cell suspension of the Escherichia coli O157H 7 cells is 107The fluorescence detection results in the cylinder microcavity structure at CFU/mL are shown in FIG. 9. As can be seen, the target cells are enriched in the microcavity structure of the channel B, and the enrichment effect is about 80% according to the statistics of the sample size.
FIG. 10 shows the results of the detection of cell suspensions of Escherichia coli O157H 7 cells at different concentrations, wherein A1-F1 are microscope brightfield images, and A2-F2 are fluorescence microscope results. The cell suspension concentrations of A to F were 10 respectively7CFU/mL、106CFU/mL、105CFU/mL、104CFU/mL、103CFU/mL、102CFU/mL. At a concentration of 102The enrichment rate is about 70% when CFU/mL is adopted, and the enrichment rate is 70% -80% under other concentrations. As can be seen from FIG. 10, the detection limit reached 100CFU/mL when the sample size was 100. mu.L.
Taking the concentration as 104And (3) capturing and enzyme-cutting the CFU/mL escherichia coli suspension by using the method, sealing the sample inlet II, applying negative pressure of 30s and vacuum degree of-0.06 Mpa to the sample outlet, and enriching the captured escherichia coli into the microcavity structure. And opening the sample inlet II, and adding a cell culture medium into the channel B of the chip from the sample inlet II so that the culture medium flows into the cylindrical microcavity structures on two sides of the channel B. The results of incubation at 37 ℃ for 0 to 18 hours are shown in FIG. 11. The result shows that the chip and the method can enrich the captured microorganisms in the microcavity structure, can perform in-vitro culture and have good growth, and the chip and the method have the advantages of small damage to target cells and capability of improving the survival rate and in-vitro amplification efficiency of in-vitro culture of the target cells.
If the direction of the fish bone on the microfluidic chip of example 1 is set to be opposite to the direction of the fluid flow, there is no significant difference in the results of capturing, enriching and detecting cells of the above examples.
If other microorganisms such as staphylococcus aureus, salmonella, vibrio parahaemolyticus, streptococcus haemolyticus and their corresponding specific aptamer sequences are used to replace escherichia coli and its aptamer sequences, the results show that the enrichment rate is over 70% and the in vitro culture growth is good.
The breast cancer CTCs, liver cancer CTCs and lung cancer CTCs and corresponding specific aptamer sequences are used for replacing escherichia coli and the aptamer sequences thereof, and the same results show that the enrichment rate is over 70 percent and the in vitro culture growth is good.
Claims (5)
1. A micro-fluidic chip for rapidly capturing or detecting cells is characterized by comprising at least one group of micro-fluidic units, wherein the number of the micro-fluidic units is 4-8, and the micro-fluidic units are arranged in an annular array;
each group of micro-fluidic units structurally comprises a channel A and a channel B which are mutually connected, wherein the channel A and the channel B are snake-shaped channels;
one end of the channel A is provided with a sample inlet I, and the sample inlet I is positioned on the outer side of the microfluidic chip; a sample inlet II is arranged at the joint of the channel A and the channel B, the tail end of the channel B is a sample outlet, and the sample outlet is positioned at the inner side of the microfluidic chip;
the depth of the channel A and the channel B is 80-200 mu m, and the width of the channel A and the channel B is 0.8-1.5 mm;
a plurality of groups of fishbone-shaped structural units are arranged in the passage A; the fishbone-shaped structural unit is positioned at the top in the linear channel of the channel A; each group of fishbone-shaped structural units comprises a plurality of fishbones which are parallel to each other, and fishbone gaps are formed between every two adjacent fishbones;
each group of fishbone-shaped structural units comprises 6-12 fishbones, the width of each fishbone is 30-50 mu m, and the depth of each fishbone is 30-50 mu m by taking the top of the channel A as a reference; in the fishbone-shaped structural units of the same group, the distance between adjacent fishbones is 80-120 mu m;
the fishbone structure comprises a fishbone micro groove A and a fishbone micro groove B, the included angles between the fishbone micro groove A and the fishbone micro groove B and the straight line channel of the channel A are respectively 30-60 degrees, and the included angle between the fishbone micro groove A and the fishbone micro groove B is 80-100 degrees; the length of the fishbone microgrooves A is not equal to that of the fishbone microgrooves B;
all fishbones are in the same or opposite direction to the flow of the fluid;
the lengths of the fishbone microgrooves A and the fishbone microgrooves B of the fishbone-shaped structural units are distributed at intervals according to the proportion of 1: 1.8-2.1 and 1.8-2.1: 1 on the same straight-line channel in the advancing direction of the fluid; on the same straight line channel, the shortest distance between adjacent fishbone-shaped structural units is 80-120 mu m;
the micro-cavity structures are arranged in the channel B and are positioned on two sides of the inner wall of the linear channel of the channel B, the micro-cavity structures are cylindrical micro-cavity structures and are sequentially distributed in an array at adjacent intervals of 80-120 microns, and the number of the cylindrical micro-cavity structures on each linear channel changes along with the length of the linear channel of the channel B; each cylindrical microcavity structure is connected to the straight channel of channel B through a small rectangular channel at the bottom,
the diameter of the cylindrical micro-cavity structure of the channel B is 80-200 μm, and the depth is 80-200 μm.
2. The microfluidic chip according to claim 1, wherein the central axis of the cylindrical microcavity structure is perpendicular to the microfluidic chip and the bottom of the linear channel of channel B, and is perpendicular to the fluid direction of the linear channel of channel B;
the diameter of the cylindrical micro-cavity structure is 100 micrometers, and the distance between adjacent cylindrical micro-cavity structures is 100 micrometers; the rectangular mini-channels have a depth of 35 μm and a width of 35 μm.
3. The microfluidic chip according to claim 1, wherein the depth of the channel a and the channel B is 100 μm, and the width is 1 mm; the diameters of the sample inlet I, the sample inlet II and the sample outlet are 1 mm; the height of the fishbone is 35 μm and the width is 35 μm based on the top of the channel A; in the fishbone-shaped structural units of the same group, the distance between adjacent fishbones is 100 mu m; on the same straight line channel, the shortest distance between adjacent fishbone-shaped structural units is 100 μm.
4. The microfluidic chip according to claim 1, wherein each group of the fishbone-shaped structural units comprises 10 fishbones, the included angle between the fishbone microgrooves A and B of the fishbones is 90 degrees, and the included angles between the fishbone microgrooves A and the fishbone microgrooves B and the two sides of the channel A are 45 degrees respectively; the lengths of the fishbone microgrooves A and the fishbone microgrooves B of the fishbone-shaped structural units are distributed at intervals in the ratio of 1:2 and 2:1 on the same straight line channel or in the advancing direction of the fluid.
5. The microfluidic chip according to claim 1, wherein the number of the microfluidic cells is 6 groups.
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| CN112553048A (en) * | 2020-12-30 | 2021-03-26 | 中国科学院长春光学精密机械与物理研究所 | Cell sorting method and chip |
| CN112553048B (en) * | 2020-12-30 | 2022-05-24 | 中国科学院长春光学精密机械与物理研究所 | Cell sorting method and chip |
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