Method for enriching and identifying circulating trophoblast cells of pregnant woman and biological chip

Abstract

The invention relates to a prenatal non-invasive detection technology for pregnant women, in particular to a method and a biochip for enriching and identifying circulating trophoblasts of pregnant women, the biochip comprises a base disc capable of centrifugally rotating, the base disc is provided with an accommodating bin, an enriching bin and an identifying bin, a micro-channel is arranged between the accommodating bin and the enriching bin, and magnetic columns are arranged on two sides of the micro-channel; a cell population rich in circulating trophoblasts and extracted from peripheral blood of a pregnant woman is added into the containing bin, the cell population flows into the micro-channel and flows into the enrichment bin after immunomagnetic beads are magnetically attracted by the magnetic column under the rotary centrifugal action, and then fluorescent staining is performed to identify the circulating trophoblasts. The method has double mechanisms of physical enrichment and immune identification, cTB can be detected from 3-5 mL of peripheral blood of a pregnant woman within one hour, and PAS-related cTB rising signals can be detected from the pregnant woman of 6-10 weeks, so that the problems of rare circulating trophoblasts, low purity and low detection efficiency in the prior art are solved.

Description

Technical Field

[0001] This invention relates to non-invasive prenatal testing technology for pregnant women, specifically a method for enriching and identifying circulating trophoblast cells in pregnant women and a biochip. Background Technology

[0002] Placenta accreta spectrum (PAS) is a group of pathological conditions in which placental villi abnormally invade the myometrium, leading to severe bleeding, uterine rupture, and maternal death. Currently, clinical diagnosis of PAS mainly relies on imaging techniques (ultrasound, MRI), but the sensitivity of imaging in early pregnancy is limited, and some cases cannot be diagnosed before delivery.

[0003] Circulating trophoblasts (cTBs) originate from the extraplacental trophoblast and are rare fetal-derived cells that enter the maternal peripheral blood circulation. Studies have shown that significantly elevated cTB levels can be detected in pregnant women with peripheral autoimmune atrophy (PAS) in early pregnancy (6–10 weeks). However, cTB concentrations in peripheral blood are extremely low (approximately 1–10 cells / mL) and are easily interfered with by leukocytes, making high-purity separation and reliable identification difficult using traditional methods. Therefore, developing a cTB enrichment and identification method combining physical separation and immunoremoval techniques to achieve rapid, accurate, and non-invasive PAS detection has significant clinical implications. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for rapidly enriching and identifying circulating trophoblast cells in pregnant women.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: a method for enriching and identifying circulating trophoblast cells in pregnant women, comprising the following steps: (1) Extraction: Collect peripheral blood samples from pregnant women and extract cell populations containing circulating trophoblasts from the samples; (2) Enrichment: Remove leukocytes from the above cell population and enrich to obtain a cell suspension; (3) Staining: Perform fluorescent staining on the above cell suspension; (4) Identification: The cells stained with the above fluorescent dyes are identified, and the cells with specific markers are identified as circulating trophoblast cells.

[0006] Preferably, an anticoagulant is first added to the collected peripheral blood sample from the pregnant woman, then the sample is spread on top of a density gradient separation solution, centrifuged, and the white membrane layer is extracted. This white membrane layer is a cell population containing circulating trophoblast cells. Alternatively, a cell lysis buffer is first added to the collected peripheral blood sample from the pregnant woman, then the sample is centrifuged, and the cell pellet is extracted. This cell pellet is a cell population containing circulating trophoblast cells.

[0007] Preferably, at least one of the above steps of enrichment, staining, and identification is completed on a biochip, or all four steps of extraction, enrichment, staining, and identification are completed on a biochip.

[0008] Preferably, immunomagnetic beads are added to the above cell population, or the above cell population is mixed with immunomagnetic beads pre-embedded on the biochip, and then leukocytes are removed by magnetic attraction.

[0009] As a preferred method, immunomagnetic beads coated with anti-CD45 antibody are used to remove leukocytes. Then, CD45, TROP2 and CEP8 markers are added to the enriched cell suspension for fluorescent staining. Cells are then identified using a fluorescence microscope, and cells that simultaneously have CD45⁻, TROP2⁺ and CEP8=2 markers are identified as circulating trophoblast cells.

[0010] As a preferred method, CD45 is used to label leukocytes, TROP2 to label trophoblast cells, and CEP8 to label fetal origin.

[0011] This invention also provides a biochip fabricated according to the above-described method for enriching and identifying circulating trophoblast cells (CTS) in pregnant women. The biochip includes a centrifugally rotatable substrate plate. At least one set of blood sample processing units is provided on the substrate plate. Each processing unit includes a receiving chamber, an enrichment chamber, and an identification chamber located on the substrate plate. A microchannel is provided between the receiving chamber and the enrichment chamber, and magnetic columns are provided on both sides of the microchannel. A cell population containing CTS extracted from a pregnant woman's peripheral blood sample is added to the receiving chamber. Under centrifugal rotation, the cell population flows into the microchannel and, after being magnetically attracted by immunomagnetic beads by the magnetic columns, flows into the enrichment chamber. Then, fluorescently labeled antibodies are added to the enrichment chamber for fluorescent staining. The fluorescently stained cell suspension flows into the identification chamber, and the identification chamber is then observed using a fluorescence microscope to identify CTS.

[0012] Preferably, the base plate has a face plate on its upper side, which has a sample compartment, an injection compartment, and a mixing compartment. A capillary microchannel is provided between the mixing compartment and the receiving compartment. Immunomagnetic beads are pre-embedded in the receiving compartment. The peripheral blood sample of the pregnant woman injected into the sample compartment and the cell extract injected into the injection compartment flow into the mixing compartment respectively to mix and obtain a cell population rich in circulating trophoblast cells. Under the siphon effect of rotational centrifugation and the capillary microchannel, the cell population flows into the receiving compartment through the capillary microchannel and mixes with the immunomagnetic beads in the receiving compartment before flowing into the microchannel.

[0013] Preferably, the face plate has a reagent well that communicates with the enrichment chamber, and fluorescently labeled antibodies added to the reagent well flow into the enrichment chamber; the face plate has a capillary microchannel inside, one end of which communicates with the lower part of the mixing chamber and the other end of which communicates with the upper part of the receiving chamber.

[0014] Preferably, the base plate includes an upper plate and a lower plate, and the face plate, upper plate, and lower plate are sealed and bonded together sequentially from top to bottom. The sample chamber, injection chamber, and reagent port are formed on the front side of the face plate, and the mixing chamber is formed on the back side of the face plate. The lower part of the sample chamber and the lower part of the injection chamber are both connected to the upper part of the mixing chamber through a liquid flow channel. The receiving chamber and enrichment chamber are formed on the front side of the upper plate. One end of the microchannel is connected to the lower part of the receiving chamber, and the other end is connected to the upper part of the enrichment chamber. Several magnetic pillars are arranged from the front and back of the upper plate toward both sides of the microchannel.

[0015] As can be seen from the above technical solutions, this invention obtains cTB through extraction, enrichment, staining, and identification steps. It combines physical enrichment and immunoassay mechanisms, enabling the detection of cTB not only from 3–5 mL of pregnant women's peripheral blood within 1 hour, but also from elevated PAS-related cTB signals in pregnant women at 6–10 weeks of gestation. This solves the problems of scarce cTB, low purity, and low detection efficiency in existing technologies. Furthermore, a biochip can be used to extract, enrich, stain, and identify cTB. Its closed structure avoids sample contamination and cell loss, and accurate cTB identification is achieved through CD45 negativity, TROP2 positivity, and two CEP8 signal points. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the front structure of the biochip of the present invention.

[0017] Figure 2 This is a partially magnified cross-sectional view of the biochip of the present invention. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0019] This invention provides a method for enriching and identifying circulating trophoblast cells in pregnant women using biochips, comprising the following steps: (1) Extraction: Collect peripheral blood samples from pregnant women and extract the cell population containing circulating trophoblast cells from the samples; for example, density gradient centrifugation can be used, specifically by first adding an anticoagulant to the collected peripheral blood samples from pregnant women, then spreading them on the upper layer of the density gradient separation solution, and then centrifuging to obtain the white film layer at the interface. This white film layer contains the cell population containing circulating trophoblast cells, that is, a cell population containing trophoblast cells, monocytes and a small number of other cells. Alternatively, cell lysis buffer can be added to the collected peripheral blood samples from pregnant women, then centrifuged and the cell pellet extracted. This cell pellet contains the cell population containing circulating trophoblast cells.

[0020] (2) Enrichment: Remove the leukocytes from the above cell population and enrich the cell suspension; specifically, add immunomagnetic beads to the above cell population, or mix the above cell population with immunomagnetic beads pre-embedded on the biochip, and then use magnetic attraction to remove leukocytes, such as using immunomagnetic beads coated with anti-CD45 antibody to remove leukocytes.

[0021] (3) Staining: Fluorescent staining is performed on the above cell suspension; specifically, CD45, TROP2 and CEP8 can be added to the enriched cell suspension for fluorescent staining, where CD45 marks leukocytes, TROP2 marks trophoblast cells, and CEP8 marks fetal origin. CD45 is used to exclude leukocytes, and cTBs must be CD45⁻. TROP2 is clearly expressed in the exochorionic trophoblast and syncytiotrophoblast lineages, and cTBs must be TROP2⁺ (membranous or cytoplasmic clearly positive). CEP8 is a chromosome 8 enumeration probe, and fetal trophoblast cells are chromosome 8 diploids. CEP8 FISH is used to confirm fetal origin, and cells must have CEP8 = 2 signal points.

[0022] (4) Identification: The cells stained with the above fluorescence are identified; if identification is performed using a fluorescence microscope, cells simultaneously marked with CD45⁻, TROP2⁺, and CEP8=2 are identified as circulating trophoblast cells. Among them, CD45⁻ indicates the absence of leukocyte marker signals, TROP2⁺ is a clear positive expression, and CEP8=2 signals indicate diploid chromosome 8. Cells that do not meet any of these criteria must be excluded. Thus, this invention uses a combination of protein markers and chromosome signals to identify cTBs, which has high specificity, can protect the integrity of cell structure, can be used in early pregnancy, and can provide risk warnings before imaging signs appear.

[0023] Furthermore, this invention defines a cTB cluster as two or more cTBs that simultaneously satisfy CD45⁻ / TROP2⁺ / CEP8=2, and aggregate in a tightly adjacent or physically connected manner; its morphological characteristics include tight connections between cells, partial fusion, or shared cytoplasm common in syncytiotrophoblasts, significantly increased overall size (often ≥30–100 μm), and enhanced TROP2 signal (due to cell membrane overlap). The number, size, and integrity of cTB clusters are closely and directly related to the depth of placental implantation, and larger or more cTB clusters are usually associated with more severe PAS stages such as increta or percreta, and can be used for non-invasive PAS staging.

[0024] In the implementation process, the above four steps of extraction, enrichment, staining, and identification can be completed without the use of a biochip. It combines physical enrichment and immunoassay mechanisms, solving the problems of scarce cTB, low purity, and low detection efficiency in existing technologies. Preferably, at least one of the enrichment, staining, and identification steps is completed on a biochip, or all four steps are completed on a biochip. Specifically: like Figure 1 and Figure 2 This invention provides a biochip fabricated according to the above-described method for enriching and identifying circulating trophoblast cells (cTB) in pregnant women. It includes a centrifugally rotatable substrate 1, on which at least one set of blood sample processing units 11 are provided (e.g., 4, 6, 8, 12 sets of processing units), thereby enabling simultaneous processing of multiple blood samples to improve processing efficiency. Each processing unit includes a receiving chamber 5, an enrichment chamber 6, and an identification chamber 10 formed on the substrate. A microchannel 12 is provided between the receiving chamber and the enrichment chamber, and magnetic columns 8 are provided on both sides of the microchannel. In practice, the substrate is placed on a centrifuge. Cells rich in cTB extracted from the peripheral blood sample of a pregnant woman are added to the receiving chamber. Under centrifugation, the cell population flows into the microchannel and, after being magnetically attracted by immunomagnetic beads by the magnetic columns, flows into the enrichment chamber. Then, fluorescently labeled antibodies are added to the enrichment chamber for fluorescent staining. The fluorescently stained cell suspension flows into the identification chamber, and the identification chamber is observed using a fluorescence microscope to identify cTB. Thus, the enrichment and identification of cTB are achieved through the biochip. This invention combines physical enrichment and immunoassay mechanisms, enabling the detection of cTB from 3–5 mL of peripheral blood within 1 hour, achieving non-invasive prenatal testing for pregnant women and serving as an early auxiliary diagnostic tool for PAS.

[0025] During implementation, the extraction of cell populations rich in circulating trophoblast cells can be achieved on the biochip of this invention. Specifically, a faceplate 13 is provided on the upper side of the base plate 1. The faceplate is provided with a sample chamber 2, an injection chamber 3, and a mixing chamber 4. A capillary microchannel 17 is provided between the mixing chamber and the containment chamber. Immunomagnetic beads 7 are pre-embedded in the containment chamber, thereby eliminating the need to add immunomagnetic beads separately and improving efficiency. The peripheral blood of the pregnant woman injected into the sample chamber and the cell extract injected into the injection chamber flow into the mixing chamber respectively for mixing to obtain a cell population rich in circulating trophoblast cells. Under the siphon effect of rotational centrifugation and the capillary microchannel, the cell population automatically flows into the containment chamber through the capillary microchannel, mixes with the immunomagnetic beads in the containment chamber, flows into the microfluidic channel, and then flows into the enrichment chamber after being magnetically attracted by the magnetic column for cTB enrichment and identification. Thus, this invention enables extraction, enrichment, and identification on a biochip, with the entire detection process taking less than 1 hour, greatly improving efficiency. Furthermore, the closed structure of the chip can prevent sample contamination and cell loss.

[0026] Preferably, the faceplate 13 has a reagent well 9 communicating with the enrichment chamber. Fluorescently labeled antibodies added to this well flow into the enrichment chamber. In this invention, CD45, TROP2, and CEP8 markers are added through the reagent well for fluorescent staining using a pipette. The faceplate contains a capillary microchannel, one end of which communicates with the lower part of the mixing chamber and the other end with the upper part of the containing chamber. This prevents cell populations from flowing into the containing chamber during mixing. The capillary microchannel of this invention consists of a first section, a middle section, and a last section. The first and last sections are inclined outwards, while the middle section is an upward-protruding arc. The inclination angle, the curvature of the arc, the length of each section, and the diameter are all calculated to ensure that the capillary microchannel functions as a capillary, enabling automatic separation of cell populations under siphon action.

[0027] The base plate 1 includes an upper plate 14 and a lower plate 15. The face plate 13, the upper plate 14 and the lower plate 15 are sealed and bonded from top to bottom. The sample chamber, the injection chamber and the reagent hole are opened on the front side of the face plate, and the mixing chamber is opened on the back side of the face plate. Thus, the mixing chamber is sealed by the sealing and bonding of the upper plate, which greatly facilitates the processing and manufacturing. The lower parts of the sample chamber and the injection chamber are connected to the upper part of the mixing chamber via a fluid channel 16, allowing peripheral blood and cell extract to flow rapidly and completely into the mixing chamber under the combined action of gravity and centrifugation. The receiving chamber and enrichment chamber are located on the front of the upper plate, and are similarly sealed by adhesive bonding to the plate. One end of the microchannel is connected to the lower part of the receiving chamber, and the other end is connected to the upper part of the enrichment chamber. This not only ensures that all cell groups in the receiving chamber flow into the enrichment chamber under centrifugation, avoiding residue, but also, because the microchannel is inclined, the pre-embedded immunomagnetic beads will not easily flow out of the receiving chamber without external force; they will only flow out under centrifugation when the specified plate speed is reached. Several magnetic pillars are arranged from the front and back of the upper plate towards both sides of the microchannel, greatly facilitating the installation of magnetic pillars and ensuring a sufficient number of pillars. The lower plate of the present invention is made of transparent material, and the identification chamber is opened from the front of the lower plate. Similarly, the identification chamber is sealed by sealing and bonding with the upper plate. The fluorescently stained cell suspension is spread flat in the identification chamber for easy optical observation.

[0028] The disc 1 of this invention is formed by sealing and bonding three layers: upper, middle, and lower. The sample chamber, injection chamber, reagent port, and mixing chamber are located in the upper layer 13, the enrichment chamber and containment chamber are located in the middle layer 14, and the identification chamber is located in the lower layer 15. This design greatly facilitates manufacturing. Specifically, the sample chamber, injection chamber, and reagent port are opened on the front side of the upper layer, and the mixing chamber is opened on the back side of the upper layer, thereby achieving a sealed mixing chamber through the sealing and bonding of the middle layer. During implementation, operations such as opening holes are performed on the front and back sides of the upper layer, making manufacturing more convenient and faster. The lower parts of the sample chamber and the lower parts of the injection chamber are connected to the upper part of the mixing chamber through the liquid flow channel 16, so that peripheral blood and cell division fluid can flow into the mixing chamber quickly and completely under the combined action of gravity and the centrifugal force of the disc rotation.

[0029] During the procedure, a cell suspension was enriched using a biochip. CD45, TROP2, and CEP8 markers were added to the enrichment chamber for fluorescent staining. Then, under the rotational centrifugation of the biochip, the fluorescently stained cell suspension automatically flowed into and spread evenly in the identification chamber. The identification chamber was then used to differentiate cells, identifying those simultaneously labeled with CD45⁻, TROP2⁺, and CEP8=2 as circulating trophoblast cells. A significantly elevated cTB count compared to normal pregnant women controls indicated a high risk of PAS, recommending further imaging confirmation, thus enabling early auxiliary diagnosis of PAS.

[0030] In the implementation process, the extraction of cell populations containing circulating trophoblast cells does not need to be achieved on the biochip of this invention. For example, after extracting the white membrane layer using density gradient centrifugation, immunomagnetic beads coated with anti-CD45 antibodies are added, and the cells are placed on a magnetic rack for static separation to remove leukocytes. The supernatant retains the CD45-negative cell population, resulting in a cell population rich in circulating trophoblast cells after removing leukocytes. This cell population is then added to the containment chamber of the biochip, and after a second leukocyte removal using a magnetic column, a high-purity cell suspension is obtained. This invention isolates mononuclear cells (PBMCs) and cTB from the white membrane layer of pregnant women's peripheral blood, and selectively removes leukocytes using magnetic beads coated with anti-CD45 antibodies. Then, automated sample separation and enrichment are achieved using the fluid sorting and magnetic separation structure within the biochip. Furthermore, leukocytes are labeled with CD45, trophoblast cells are labeled with TROP2, and fetal-derived cells are labeled with CEP8, thereby specifically identifying cTB. This solves the problems of scarce circulating trophoblast cells, low purity, and low detection efficiency in existing technologies.

[0031] In the extraction of cell populations rich in circulating trophoblast cells (cTB) on the biochip of the present invention, as a preferred method, peripheral blood from the pregnant woman injected into the sample chamber of the biochip and density gradient separation solution injected into the injection chamber are first mixed in a mixing chamber and then separated into layers by centrifugation. The separated white membrane layer is then automatically extracted into a receiving chamber using a capillary microchannel to obtain a cell population rich in cTB. The cell suspension is then enriched. Thus, the entire process of cTB extraction, enrichment, and identification is achieved on the biochip of the present invention using density gradient centrifugation. During the implementation, a separation solution with a density of 1.077 g / mL, such as Ficoll or its economical alternative, is used to separate and centrifuge the anticoagulated peripheral blood to obtain a white membrane layer rich in trophoblast cells. Immunomagnetic beads coated with anti-CD45 antibodies are then mixed and incubated with the white membrane layer cells. Leukocytes are removed by magnetic adsorption, retaining CD45-negative cTB. This method combines gradient centrifugation with immunoleukogenesis to remove maternal cell background to the greatest extent, and achieves accurate cTB determination through CD45⁻ / TROP2⁺ / CEP8=2 labeling.

[0032] As another preferred method, the peripheral blood sample from the pregnant woman injected into the sample compartment of the biochip and the cell lysis buffer injected into the injection compartment are separately flowed into a mixing compartment for mixing and lysis. Then, under centrifugal rotation, the lysed cell pellet is automatically extracted into a receiving compartment using capillary microchannels to obtain a cell population rich in circulating trophoblast cells, and the cell suspension is then enriched. This method uses cell lysis and pre-embedded immunomagnetic beads on the biochip, thereby achieving automatic transfer of cell pellet, separation of immunomagnetic beads, and enrichment of cTB under centrifugal rotation. This saves steps, further improves efficiency, and provides a novel non-invasive solution for the rapid diagnosis of PAS, which will significantly improve the clinical management pathway for PAS. Example

[0033] 3-5 mL of peripheral blood samples were collected from pregnant women, and an appropriate amount of anticoagulant was added. The sample was slowly spread on the upper layer of a gradient density separation solution with a density of 1.077 g / mL, and centrifuged at 400g for 20 min to achieve stratification. The white membrane layer at the interface was collected, and immunomagnetic beads coated with anti-CD45 antibody were added. After incubation at room temperature for 5-15 min, the sample was placed on a magnetic rack for static separation. After magnetic aspiration for 3-5 min, the leukocytes bound to the immunomagnetic beads were removed, and the CD45-negative cell population in the supernatant was retained. The cell population was then added to the containment chamber of the biochip and leukocytes were removed a second time by a magnetic column to obtain a high-purity cTB cell suspension. Then, CD45, TROP2, and CEP8 markers were added to the enrichment chamber for fluorescent staining, and the cells were spread evenly in the identification chamber. The specific number of cells with CD45⁻ / TROP2⁺ / CEP8=2 was observed using a fluorescence microscope. The entire process lasted less than 1 hour. During observation, a comprehensive judgment is made based on the marker expression pattern, fluorescence intensity, nuclear morphology, CEP8 FISH signal number and distribution, and whether clusters are formed. Cluster structures can be confirmed manually or automatically identified by software.

Claims

1. A method for enriching and identifying circulating trophoblast cells in pregnant women, characterized in that... Includes the following steps: (1) Extraction: Collect peripheral blood samples from pregnant women and extract cell populations containing circulating trophoblasts from the samples; (2) Enrichment: Remove leukocytes from the above cell population and enrich to obtain a cell suspension; (3) Staining: Perform fluorescent staining on the above cell suspension; (4) Identification: The cells stained with the above fluorescent dyes are identified, and the cells with specific markers are identified as circulating trophoblast cells.

2. The method for enriching and identifying circulating trophoblast cells in pregnant women according to claim 1, characterized in that: First, an anticoagulant is added to the collected peripheral blood sample from the pregnant woman, then the sample is spread on top of a density gradient separation solution, centrifuged, and the white membrane layer is extracted. This white membrane layer is a cell population containing circulating trophoblast cells. Alternatively, cell lysis buffer is added to the collected peripheral blood sample from the pregnant woman, then the sample is centrifuged, and the cell pellet is extracted. This cell pellet is a cell population containing circulating trophoblast cells.

3. The method for enriching and identifying circulating trophoblast cells in pregnant women according to claim 1, characterized in that: At least one of the above steps of enrichment, staining, and identification is completed on a biochip, or all four steps of extraction, enrichment, staining, and identification are completed on a biochip.

4. The method for enriching and identifying circulating trophoblast cells in pregnant women according to claim 1, characterized in that: Immunomagnetic beads are added to the above cell population, or the above cell population is mixed with immunomagnetic beads pre-embedded on a biochip, and then leukocytes are removed by magnetic attraction.

5. The method for enriching and identifying circulating trophoblast cells in pregnant women according to claim 4, characterized in that: Leukocytes were removed using immunomagnetic beads coated with anti-CD45 antibody. Then, CD45, TROP2 and CEP8 markers were added to the enriched cell suspension for fluorescent staining. Cells were then identified using a fluorescence microscope. Cells simultaneously labeled with CD45⁻, TROP2⁺ and CEP8=2 were identified as circulating trophoblast cells.

6. The method for enriching and identifying circulating trophoblast cells in pregnant women according to claim 5, characterized in that: CD45 is used to label leukocytes, TROP2 to label trophoblast cells, and CEP8 to label fetal origin.

7. A biochip fabricated using the method for enriching and identifying circulating trophoblast cells in pregnant women according to any one of claims 1 to 6, comprising a centrifugally rotatable substrate disc, wherein the substrate disc is provided with at least one set of blood sample processing units, characterized in that: Each processing unit includes a containment chamber, an enrichment chamber, and a differentiation chamber located on the substrate. A microchannel is provided between the containment chamber and the enrichment chamber, and magnetic columns are provided on both sides of the microchannel. A cell population containing circulating trophoblast cells extracted from a pregnant woman's peripheral blood sample is added to the containment chamber. Under the action of rotational centrifugation, the cell population flows into the microchannel and then flows into the enrichment chamber after being magnetically attracted to immunomagnetic beads by the magnetic columns. Then, fluorescently labeled antibodies are added to the enrichment chamber for fluorescent staining. The fluorescently stained cell suspension flows into the differentiation chamber, and the differentiation chamber is then observed using a fluorescence microscope to identify circulating trophoblast cells.

8. The biochip according to claim 7, characterized in that: The base plate has a face plate on its upper side, which has a sample compartment, an injection compartment, and a mixing compartment. A capillary microchannel is provided between the mixing compartment and the receiving compartment. Immunomagnetic beads are pre-embedded in the receiving compartment. The peripheral blood sample of the pregnant woman injected into the sample compartment and the cell extract injected into the injection compartment flow into the mixing compartment respectively to mix and obtain a cell population rich in circulating trophoblast cells. Under the siphon effect of rotational centrifugation and the capillary microchannel, the cell population flows into the receiving compartment through the capillary microchannel and mixes with the immunomagnetic beads in the receiving compartment before flowing into the microchannel.

9. The biochip for enriching and identifying circulating trophoblast cells in pregnant women according to claim 8, characterized in that: The faceplate has a reagent well that communicates with the enrichment chamber, and fluorescently labeled antibodies added to the reagent well flow into the enrichment chamber; the faceplate has a capillary microchannel inside, one end of which communicates with the lower part of the mixing chamber and the other end of which communicates with the upper part of the receiving chamber.

10. The biochip for enriching and identifying circulating trophoblast cells in pregnant women according to claim 9, characterized in that: The base plate includes an upper plate and a lower plate. The upper plate, the lower plate, and the base plate are sealed and bonded together from top to bottom. The sample chamber, the injection chamber, and the reagent port are formed on the front side of the base plate, and the mixing chamber is formed on the back side of the base plate. The lower parts of the sample chamber and the injection chamber are connected to the upper part of the mixing chamber through a liquid flow channel. The receiving chamber and the enrichment chamber are formed on the front side of the upper plate. One end of the microchannel is connected to the lower part of the receiving chamber, and the other end is connected to the upper part of the enrichment chamber. Several magnetic pillars are arranged from the front and back of the upper plate toward both sides of the microchannel.

Images