CN115999660A - Microfluidic chip and application thereof - Google Patents

Abstract

The invention provides a microfluidic chip suitable for large-scale continuous separation of target particles, such as circulating tumor cells, and applications thereof, such as hemodialysis.

Description

Microfluidic chip and application thereof

Incorporation by reference

The application requires that the application number of the China national intellectual property agency is 202111124445.7, the application name is 'a microfluidic chip' and the priority of the China application of the microfluidic chip is submitted 24 days of 2021. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference and set forth in its entirety herein.

Technical Field

The invention belongs to the field of biomedical engineering, and particularly relates to a microfluidic chip and application thereof in removing, separating and/or enriching target particles (such as circulating tumor cells).

Background

Most cancer patients (> 95%) have metastasized at the time of diagnosis, with metastasis being the leading cause of cancer death, and more than 90% of cancer patients die from metastasis rather than primary tumors. Current mainstream cancer treatments (surgery, radiation and chemotherapy) are not prescribed. The 5-year survival rate has been scarcely improved in cancer patients who have developed metastasis for the past 10 years. Cancer cells shed from the carcinoma in situ, pass through the interstices between vascular endothelial cells and enter the blood as circulating tumor cells (circulating tumor cell, CTCs). CTCs are a key loop in cancer metastasis, and no CTCs have no metastasis of cancer. Numerous studies have shown that CTCs can be detected in the blood well before cancer forms metastases. In addition, the number of CTCs is closely related to patient prognosis, and the total survival (22.6 months) of patients with a CTC number of 5/7.5 mL is much lower than that of patients with a CTC number of < 5/7.5 mL (4.1 months). Therefore, by removing CTCs in blood or reducing the number of CTCs in blood by means of dialysis, the progress of cancer metastasis can be blocked, and the prognosis of the patient can be significantly improved.

Although there are a number of methods available to achieve separation and capture of CTCs, there is a large distance from true CTC dialysis. CTC capture based on antigen-antibody requires antibody coating in advance, and the operation process is complicated and the cost is high. More importantly, the flow rate that it can be used is low, does not match the blood flow rate, and has a limited capture site. The method (1) existing at present

The Seldinger wire was coated with EpCAM antibody and then inserted into the elbow vein of a cancer patient by way of an indwelling needle for 30min, and CTCs captured on the wire were immunofluorescent stained and detected. (2) By coating the resistAnd the CTC capturing chip of the body is connected with a microcontroller, a peristaltic pump and a heparin syringe to capture and detect CTC in canine model venous blood. With this method, 10-20mL of blood (1% -2% of the total blood volume) can be analyzed for 2 hours. In addition, there are CTC separation methods based on physical properties, but there are also many problems such as: (1) Flow rate mismatch, often too high or too low, and (2) blood often requires pretreatment, such as large scale dilution or lysis of red blood cells, resulting in the inability of the outlet-derived blood cells to be directly fed back without damage.

In this case, there is also a need for alternative methods and devices for isolating/removing/capturing CTCs at both the analytical and preparative level to meet the diagnostic and therapeutic needs of cancer patients.

Disclosure of Invention

The inventors have previously proposed a microfluidic chip suitable for capturing circulating tumor cells, which is described in chinese patent application nos. 201910666239.5 (published as CN111909828 a) and 202022766020.3, the entire disclosures of which are incorporated herein by reference.

Based on the above, the inventor provides a system for separating, qualitatively and quantitatively detecting tumor cells in body circulation based on a microfluidic chip through a great deal of researches. The system can separate and remove tumor cells in blood circulation, can slow or block cancer metastasis, and improve prognosis. In addition, by detecting and analyzing the separated circulating tumor cells, personalized real-time information of multiple groups of cancers can be obtained, and the purposes of accurate diagnosis, drug screening, efficacy evaluation, prediction prognosis, recurrence monitoring and the like are achieved. In addition, more advanced research (including single cell sequencing and the like) on CTC can lay a foundation for deep understanding of the mechanism of cancer occurrence and metastasis and searching for better clinical treatment targets. In addition, the system is also suitable for processing large volumes of other body fluids (e.g., urine, ascites, lavage, leukopenia enrichment, etc.), from which CTCs can be obtained using the system of the present invention, except that only no reinfusion can be collected.

In a first aspect, the present invention provides a microfluidic chip comprising a main inlet (1), a main channel (2), one or more (e.g. 1-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) converging and diverging units (3), a collecting port (4) and a diverging port (5) arranged in series, wherein the main inlet (1), the one or more converging and diverging units (3) and the collecting port (4) are connected by and in fluid communication with the main channel (2);

wherein the chip is configured such that fluid enters the main channel (2) via the main inlet (1) and flows through the one or more converging and diverging units (3), each converging and diverging unit (3) converges target particles in the fluid at the center of the fluid flow, the fluid rich in target particles enters the latter converging and diverging unit or flows out via the collecting port (4), and the fluid with reduced content of target particles flows out from the diverging port (5).

In a second aspect, the present invention provides a device for separating, enriching, concentrating, collecting, removing and/or analysing particles of interest, comprising a microfluidic chip according to the first aspect of the invention. In one embodiment, the device is a dialysis device.

In a third aspect, the present invention provides a method of separating, enriching, concentrating, collecting, removing and/or analyzing target particles comprising:

(1) Providing a fluid containing or to be determined whether to contain target particles, and

(2) The fluid is caused to flow through a microfluidic chip according to the first aspect of the invention or a device according to the second aspect of the invention.

In one embodiment, the method is a dialysis method comprising

(1) Providing a fluid to be dialyzed, wherein the fluid to be dialyzed comprises or is suspected to comprise target particles,

(2) Flowing the fluid through a microfluidic chip according to the first aspect of the invention or a dialysis device according to the second aspect of the invention, and

(3) The fluid from the shunt port with reduced target particle content is collected.

In a specific embodiment of the invention, the fluid is selected from the group consisting of blood, plasma, serum, perfusion fluid, ascites, leukopenia enrichment fluid, urine, tissue fluid, cerebrospinal fluid, cell culture fluid and cell mixtures, more specifically from the group consisting of blood, perfusion fluid, ascites and leukopenia enrichment fluid, such as blood.

In a specific embodiment of the invention, the target particle is a tumor cell, preferably a circulating tumor cell.

The microfluidic chip and the system for separating, qualitatively and quantitatively detecting the tumor cells in the systemic circulation, provided by the invention, have at least one of the following beneficial effects when blood samples are processed:

1. the separation of CTC is carried out by adopting a method based on physical properties, the flow rate can be matched with the flow rate of blood in a human body, the blood can not be too high or too low, and the blood with larger volume (tens to thousands of mL/h) can be processed in a shorter time;

2. the blood does not need pretreatment and can be directly introduced into the microfluidic chip;

3. the total volume of the internal channels of the microfluidic chip is small, and the volume of blood which is finally retained in the chip is small, so that the damage is reduced;

4. the whole process only needs one step and one fixed flow rate, so that the separation of CTC and other blood cells can be realized, and the method is simple and convenient;

5. the blood cells obtained from the outlet can be directly returned to the body.

The microfluidic chip and the system for separating, qualitatively and quantitatively detecting the tumor cells in the systemic circulation can separate and remove the tumor cells in the blood circulation, can slow down or block the metastasis of the cancer, and improve the prognosis. Furthermore, by detecting and analyzing the separated circulating tumor cells, personalized real-time information of multiple groups of cancers can be obtained, and the purposes of accurate diagnosis, drug screening, efficacy evaluation, prediction prognosis, recurrence monitoring and the like are achieved. In addition, the further research on CTC can lay the foundation for the deep understanding of the mechanism of cancer occurrence and metastasis and the search of better clinical treatment targets.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1A is a design diagram of a microfluidic chip including 4 groups of converging and diverging units, and fig. 1B is a physical diagram of a correspondingly fabricated chip;

FIG. 2 is similar to FIG. 1A, except that each set of aggregate branches in FIG. 1A uses its own split port, while the common branch bus in FIG. 2 is connected to a unified split port;

FIG. 3 is a design drawing of a group of converging and diverging units comprising 5 converging structures;

FIG. 4 is a schematic diagram of a microfluidic chip comprising 16 sets of converging and diverging units;

FIG. 5 is similar to FIG. 4, except that each set of aggregate branches in FIG. 4 uses its own split, whereas the shared split bus in FIG. 5 is connected to a unified split;

FIG. 6 is a schematic diagram of a microfluidic chip containing 16 sets of converging and diverging units and containing a filter structure prior to entry;

FIG. 7 is similar to FIG. 6, except that each set of aggregate branches in FIG. 6 uses its own split, whereas the shared split bus in FIG. 7 is connected to a unified split;

fig. 8A is a design drawing of a microfluidic chip including 4 groups of converging and diverging units and including a filter structure before inlet, and fig. 8B is a physical diagram of a correspondingly fabricated chip;

fig. 9A is a design diagram of a microfluidic chip comprising two cascaded converging and diverging units connected in series, and fig. 9B is a physical diagram of a correspondingly fabricated chip;

fig. 10A is a design diagram of a microfluidic chip comprising two cascaded converging and diverging units connected in parallel, and fig. 10B is a physical diagram of a correspondingly fabricated chip;

FIG. 11 is a schematic diagram of a separation system based on a microfluidic chip of the present invention;

FIG. 12 is a schematic view of a hemodialysis apparatus based on a microfluidic chip of the present invention;

fig. 13 is a graph showing statistics of capture efficiency of CTCs filtered using a mouse model.

Detailed Description

Other aspects and advantages of the present invention will become apparent to those skilled in the art upon review of the following detailed description. It is noted that the following detailed description (including the drawings) is merely exemplary and is not limiting of the invention. It will be readily understood that the present invention may be carried out in other and different embodiments by modifying and changing some specific details without departing from the invention.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is clear that the described examples are only illustrative and not exhaustive of all the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.

Features described in the various embodiments described herein may be combined in accordance with the spirit of the invention and these combined embodiments are also part of the disclosure herein and are within the scope of the invention.

Unless otherwise indicated, terms used herein have the ordinary meaning as understood by one of ordinary skill in the art to which they pertain.

As used herein, unless expressly stated otherwise, a term is not preceded by an article or by the modification of "the" meaning that the term is one or more.

The term "about" or "approximately" generally means within the error range of a particular value determined by one of ordinary skill, depending in part on the manner of measurement, i.e., limited by the measurement system. For example, according to particular field practices, "about" may refer to one or more standard deviations. In particular, "about" may refer to a value within a range of 20%, 10%, 5%, or 1% of a given value deviation.

In a first aspect, the present invention provides a microfluidic chip comprising a main inlet (1), a main channel (2), one or more (e.g. 1-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) converging and diverging units (3), a collecting port (4) and a diverging port (5) arranged in series, wherein the main inlet (1), the one or more converging and diverging units (3) and the collecting port (4) are connected by the main channel (2) and are in fluid communication;

wherein the chip is configured such that fluid enters the main channel (2) via the main inlet (1) and flows through the one or more converging and diverging units (3), each converging and diverging unit (3) converges target particles in the fluid at the center of the fluid flow, the fluid rich in target particles enters the latter converging and diverging unit or flows out via the collecting port (4), and the fluid with reduced content of target particles flows out from the diverging port (5).

In one embodiment, each of the converging splitting units (3) comprises a secondary inlet (31), one or more (e.g. 1-20, more e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) converging structures (32) arranged in series, and a splitting channel (33);

wherein the converging and diverging units are configured such that fluid flowing in from the main inlet (1) or the front converging and diverging unit enters the one or more converging structures (32) arranged in series via the secondary inlet (31), each converging structure (32) converges target particles in the fluid at the center of the fluid flow, and fluid rich in target particles enters the rear converging structure or the rear converging and diverging unit, or flows out via the collecting port (4), and fluid with reduced content of target particles enters the diverging port (5) via the diverging channel (33).

In another embodiment, each of the converging structures (32) includes a central passage (321) and a side branch passage (322); wherein the central channel is connected with the main channel at both ends; the side branch passages are located beside (e.g., on one or both sides of) the central passage and meet the main passage and the central passage at both ends.

In another embodiment, the shunt channels (33) are located downstream of the final converging structure and distributed beside (e.g., on one or both sides of) the main channel.

In another embodiment, the fluid with reduced target particle content in each converging splitting unit of the series arrangement enters the common splitting bus (34) via the respective splitting channel (33) and is released via the common splitting port (5), or

The fluid with reduced content of target particles in each converging and diverging unit of the series arrangement enters the respective diverging port (5) via the respective diverging channel (33) for release.

In another embodiment, the microfluidic chip further comprises a filtration unit (6) located before the first stage converging and splitting unit such that fluid entering the main inlet flows through the filtration unit before entering the converging and splitting unit.

In another embodiment, the microfluidic chip does not comprise a capture unit, but is configured such that the fluid enriched in target particles flows out of the final converging and diverging unit directly via the collection port (4).

In another embodiment, the one or more series-arranged aggregate-shunt units are referred to as an aggregate-shunt unit cascade, and the chip comprises 2 or more (e.g., 2-2000 or 2-10000, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 10000 or more) of the aggregate-shunt unit cascade arranged in parallel.

In a specific embodiment, the microfluidic chip comprises a multi-layer structure (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more layers), in particular each layer of the multi-layer structure comprises 1 or more cascades of the converging and diverging units arranged in parallel, and the cascades of the converging and diverging units of each layer are connected in parallel, in series or both in parallel and in series.

In a second aspect, the present invention provides a device for separating, enriching, concentrating, collecting, removing and/or analysing particles of interest, comprising a microfluidic chip according to the first aspect of the invention. In one embodiment, the device is a dialysis device. In a specific embodiment, the device further comprises a filtration unit (6) located upstream of and independent of the microfluidic chip.

In a third aspect, the present invention provides a method of separating, enriching, concentrating, collecting, removing and/or analyzing target particles comprising:

(1) Providing a fluid containing or to be determined whether to contain target particles, and

(2) The fluid is caused to flow through a microfluidic chip according to the first aspect of the invention or a device according to the second aspect of the invention.

In one embodiment, the method is a dialysis method comprising

(1) Providing a fluid to be dialyzed, wherein the fluid to be dialyzed comprises or is suspected to comprise target particles,

(2) Flowing the fluid through a microfluidic chip according to the first aspect of the invention or a dialysis device according to the second aspect of the invention, and

(3) The fluid from the shunt port with reduced target particle content is collected.

In a specific embodiment, the method further comprises

(4) The fluid enriched in target particles from the collection port is collected.

In the present invention, the fluid may be of various sources, for example the fluid is selected from the group consisting of blood, plasma, serum, perfusion fluid, ascites, leukopenia enrichment fluid, urine, tissue fluid, cerebrospinal fluid, cell culture fluid and cell mixtures, more particularly from the group consisting of blood, perfusion fluid, ascites and leukopenia enrichment fluid, for example blood.

As used herein, the target particles may also be different types of particles, for example tumor cells, preferably Circulating Tumor Cells (CTCs).

Examples

Example 1 microfluidic chip fabrication

The fabrication of a conventional microfluidic chip can be referred to as: [1] lu C, xu J, han J, et al, A novel microfluidic device integrating focus-separation speed reduction design and trap arrays for high-throughput capture of circulating tumor cells [ J ]. Lab on a Chip,2020,20 (22): 4094-4105 [2]Khoo,Bee,Luan,et al.Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device [ J ]. Nature protocols erecipes for researchers,2018 [3]Tian W,Shao C,Li L,et al.Streamline-based purification of bacterial samples from liquefied sputum utilizing microfluidics [ J ]. Lab on A Chip,2017,17 (21).

In the invention, the micro-fluidic chip can be manufactured by referring to the embodiment 1 of Chinese patent application publication No. CN111909828A, and aiming at a specific separation target, a mask (1) is manufactured and a graph is drawn by using L-Edit software; (2) And exposing the chromium plate by using a laser direct-writing instrument UPG-501, and developing by using a developing solution to form a mask. Manufacturing a die (3) and baking a silicon wafer/chromium plate: taking a silicon wafer/chromium plate with proper size, placing the smooth surface of the silicon wafer/chromium plate upwards, and heating on an electric heating plate at 190 ℃ for 5min to remove water molecules attached to the surface; and (4) spin coating: selecting the type of SU-8 photoresist and the spin speed of photoresist according to the required thickness and referring to a photoresist-homogenizing curve; and (5) pre-baking: the silicon wafer/the chromium plate after the spin coating is taken out and is placed on an electric heating plate at the temperature of 95 ℃ for heating for a certain time; (6) exposure: placing a chromium plate above the uniformly glued silicon wafer, and striking a mercury lamp on the chromium plate for exposure; and (7) post-baking: the exposed silicon wafer is placed on an electric heating plate at the temperature of 95 ℃ again for heating for a certain time. (8) developing: and (3) soaking the silicon wafer in a developing solution, sucking the pure developing solution by using a disposable liquid transferring pipe, repeatedly flushing, cleaning the photoresist, and drying by using a nitrogen gun or an ear washing ball. Preparation of PDMS chip (9) PDMS A and B were glued in a ratio of 7:1-10:1 are mixed and stirred in a culture dish; (10) Pouring the uniformly mixed PDMS on a prepared mould, and vacuumizing to remove bubbles; (11) The mold together with PDMS was then placed in an oven at 75 ℃ and heat cured for about 1 hour. (12) PDMS was cut out, punched, and bonded to a slide using a PLASMA cleaner. After bonding, it is necessary to place the material in an oven at 75℃overnight for reinforcement. Specifically, the inventors fabricated a variety of microfluidic chips of the present invention, see for example fig. 1A and 1B, fig. 8A and 8B, fig. 9A and 9B, and fig. 10A and 10B.

Example 2 microfluidic chip 1

Referring to fig. 1A, a specific microfluidic chip was designed. The chip comprises a main inlet 1, a main channel 2, 4 converging and splitting units 3, a collecting port 4 and a splitting port 5 which are arranged in series, wherein the main inlet 1, the 4 converging and splitting units 3 and the collecting port 4 which are arranged in series are connected by the main channel 2 and are in fluid communication, each converging and splitting unit 3 is respectively provided with a splitting channel 33 towards two sides, and the tail end of each splitting channel is respectively provided with a splitting port 5, so that fluid without target particles in each converging and splitting unit enters the respective splitting port 5 through the respective splitting channel 33 to be released.

Referring to fig. 3, each converging splitter unit 3 includes a secondary inlet 31, a plurality of converging structures 32 arranged in series (e.g., 5 converging structures are shown in fig. 3), and a splitting channel 33. Each of the convergence structures 32 includes a central passage 321 and a side branch passage 322; wherein the central channel is connected with the main channel at both ends; the side branch passages are positioned at two sides of the central passage and are intersected with the main passage and the central passage at two ends. The split channels 33 are located downstream of the final converging structure and are distributed on both sides of the main channel.

The whole converging and diverging unit is configured such that fluid flowing in from the main inlet 1 or the front converging and diverging unit enters the plurality of converging structures 32 arranged in series via the secondary inlet 31, each of the converging structures 32 converges target particles in the fluid at the center of the fluid flow, and fluid rich in target particles enters the rear converging structure or the rear converging and diverging unit, or flows out via the collecting port 4, and fluid with reduced content of target particles enters the diverging port 5 via the diverging passage 33.

The whole chip is designed so that fluid to be separated enters the main channel 2 through the main inlet 1 and flows through the plurality of converging and splitting units 3 which are arranged in series, each converging and splitting unit 3 converges target particles in the fluid to be separated at the center of liquid flow, the fluid rich in the target particles enters the rear-stage converging and splitting unit or flows out through the collecting port 4, and the fluid with reduced content of the target particles flows out through the splitting port 5.

Referring to fig. 1B, this is a microfluidic chip actually fabricated according to the design of fig. 1A using the method of example 1, in which holes are punched in the chip surface to form a main inlet 1, a collection port 4, and a plurality of shunt ports 5. The parameters of the microfluidic chip can be adjusted according to the application scenario. Specific values for these parameters can be readily determined by one skilled in the art based on the teachings herein and the actual needs. As an example, the main passage 2 has a diameter of 90 μm, the branch passage 33 has a diameter of 60 μm, the center passage 321 has a diameter of 30 μm, and the side branch passage 322 has a diameter of 30 μm.

Example 3 microfluidic chip 2

Referring to fig. 2, a specific microfluidic chip was designed. The chip is similar to the chip described in example 2 (see fig. 1A) and comprises a main inlet 1, a main channel 2, 4 converging and diverging units 3 arranged in series, a collecting port 4 and a diverging port 5, wherein the main inlet 1, the 4 converging and diverging units 3 arranged in series and the collecting port 4 are connected by the main channel 2 and are in fluid communication, and a diverging channel 33 is respectively arranged on two sides of each converging and diverging unit 3. In fig. 1A, a diversion port 5 is provided at the end of each diversion channel, so that the fluid with reduced content of target particles in each converging diversion unit enters the respective diversion port 5 via the respective diversion channel 33 to be released; in contrast, in fig. 2, each diverter channel distally merges into a common diverter bus 34 and connects to a respective diverter port 5, and fluid of reduced target particle content in each converging diverter unit enters the common diverter bus 34 via a respective diverter channel 33 and is released via the common diverter port 5. Parameters of the microfluidic chip can be adjusted according to the application scene. Specific values of the chip parameters can be readily determined by one skilled in the art based on the present disclosure and actual needs. As an example, the main passage 2 has a diameter of 180 μm, the split passage 33 has a diameter of 60 μm, the center passage 321 has a diameter of 60 μm, and the side branch passage 322 has a diameter of 45 μm.

Example 4 microfluidic chip 3

Referring to fig. 4, a specific microfluidic chip was designed. The chip is similar to the chip described in example 2 (see fig. 1A), except that 4 series-arranged converging and diverging units 3 are provided in the microfluidic chip of fig. 1A, and 16 series-arranged converging and diverging units 3 are provided in the microfluidic chip of fig. 4.

Example 5 microfluidic chip 4

Referring to fig. 5, a specific microfluidic chip was designed. The chip is similar to the chip described in example 3 (see fig. 2), except that 4 series-arranged converging and diverging units 3 are provided in the microfluidic chip of fig. 2, and 16 series-arranged converging and diverging units 3 are provided in the microfluidic chip of fig. 5.

Example 6 microfluidic chip 5

Referring to fig. 6, a specific microfluidic chip was designed. The chip is similar to the chip described in example 4 (see fig. 4), except that the pre-filter unit 6 is not provided in the microfluidic chip of fig. 4, whereas the pre-filter unit 6 is provided before the first-stage converging and diverging unit in the microfluidic chip of fig. 6.

Example 7 microfluidic chip 6

Referring to fig. 7, a specific microfluidic chip was designed. The chip is similar to the chip described in example 5 (see fig. 5), except that in the microfluidic chip of fig. 5 no pre-filter unit 6 is provided, whereas in the microfluidic chip of fig. 7 a pre-filter unit 6 is provided before the first stage converging and diverging unit.

Example 8 microfluidic chip 7

Referring to fig. 8A, a specific microfluidic chip was designed. The chip is similar to the chip described in example 2 (see fig. 1A), except that 4 converging and diverging units 3 are provided and a pre-filter unit 6 is not provided in the microfluidic chip of fig. 1A; in the microfluidic chip of fig. 8A, 4 converging and diverging units 3 are provided, and a pre-filter unit 6 is provided before the first converging and diverging unit. Fig. 8B shows a microfluidic chip actually fabricated using the method of example 1 according to the design of fig. 8A.

Example 9 microfluidic chip 8

Referring to fig. 9A, a specific microfluidic chip was designed. The chip is similar to the chip described in example 8 (see fig. 8A), except that the microfluidic chip of fig. 8A only comprises 1 cascade of converging and diverging units, whereas the microfluidic chip of fig. 9A comprises two cascades of converging and diverging units arranged in series on a single plane. Fig. 9B shows a microfluidic chip actually fabricated using the method of example 1 according to the design of fig. 9A.

Example 10 microfluidic chip 9

Referring to fig. 10A, a specific microfluidic chip is designed. The chip is similar to the chip described in example 8 (see fig. 8A), except that the microfluidic chip of fig. 8A includes only 1 cascade of converging and diverging units, while the microfluidic chip of fig. 10A includes two cascades of converging and diverging units arranged in parallel on a single plane. Fig. 10B shows a microfluidic chip actually fabricated using the method of example 1 according to the design of fig. 10A.

Example 11 microfluidic chip-based separation System

Fig. 11 shows a separation system based on the microfluidic chip of the present invention. The separation system comprises an injection pump device, a microfluidic chip and a microscope system (comprising an objective lens, an objective table, a mercury lamp, an optical filter and a CCD (charge coupled device)) which are arranged in the injection pump device. The blood is pushed into the microfluidic chip by using a syringe pump, the liquid rich in Circulating Tumor Cells (CTC) is gradually enriched and concentrated, the liquid is collected from a collection port, and the liquid with reduced/eliminated CTC flows out from a shunt port. The CTC-rich liquid collected was imaged using dedicated marker staining and photographed and counted using a microscope.

Example 12 hemodialysis device and in-vivo CTC separation and detection System based on microfluidic chip

Fig. 12 shows a hemodialysis device based on the microfluidic chip of the present invention, which can be further used as an in-vivo CTC separation and detection system. The dialysis device/in-vivo CTC separation and detection system comprises two parts: CTC enrichment and separation module (including microfluidic chip 902, microfluidic chip connection conduits 921 and 922), and blood extracorporeal circulation module (including controllable connection valves 911 and 912, arteriovenous connection conduits 915 and 916, peristaltic pump 913, and heparin pump 914). The controllable connection valves 911 and 912 are used to connect the arteriovenous connection conduits 915 and 916 and the microfluidic chip connection conduits 921 and 922 while controlling communication and closure between the conduits. The arterial or venous connection catheter is used for connection of the living animal/patient to an external instrument device, and the peristaltic pump 913 is used to effect blood flow from the living animal/patient to the microfluidic chip 902. The peristaltic pump may be used to control the rate of blood flow from the living animal/patient to the microfluidic chip; the heparin pump 914 is used to achieve heparin instillation of blood to prevent clotting throughout the dialysis process. The device/system further comprises a microscope 903 and a control computer 904.

The CTC enrichment and separation module is used for enriching and separating circulating tumor cells and circulating tumor cell clusters in the blood of living animals/patients; and the blood extracorporeal circulation module is used for establishing connection between the CTC enrichment and separation module and the living animal/patient, so that the sample injection of blood to the CTC enrichment and separation module and the reinfusion of blood flowing out of the CTC enrichment and separation module to the living animal/patient are realized.

The materials used for the microfluidic chip connecting conduits 921 and 922 are not limited as long as the objects of the present invention can be achieved, and for example, materials having good biocompatibility can be used, and specifically, materials can be selected from but not limited to silica gel type or polyurethane type conduits. The inner diameter of the catheter is not limited as long as the object of the present invention can be achieved, and may be, for example, 0.1mm to 6cm; the outer diameter is not limited, and may be, for example, 0.1mm to 6cm.

The material of the arterial or venous connection catheters 915 and 916 is not limited as long as the object of the present invention can be achieved, and for example, a silicone-based catheter or a polyurethane catheter having superior biocompatibility can be used. The inside diameter of the arterial or venous connection catheter is not limited as long as the object of the present invention can be achieved, and may be, for example, 0.1mm to 6cm; the outer diameter is not limited, and may be, for example, 0.1mm to 6cm.

The arterial or venous connection catheters 915 and 916 are collectively referred to as arterial connection catheter 915 and venous connection catheter 916. The microfluidic chip connection conduits 921 and 922 include a conduit 921 connected to an inlet of the microfluidic chip and a conduit 922 connected to an outlet of the microfluidic chip. Wherein, one end of the arterial connecting conduit 915 is used for connecting with the artery of the living animal/patient, and the other end is connected with the conduit 921 connected with the inlet of the microfluidic chip; the venous connection tube 916 has one end for connection to a vein of a living animal/patient and the other end connected to a tube 922 connected to an outlet of the microfluidic chip.

Further, the peristaltic pump 913 is disposed on the arterial connection catheter.

Further, the heparin pump 914 may be directly connected to a vein of a living animal/patient through a catheter for instilling heparin into the living animal/patient.

The blood of living animals/patients flows into the microfluidic chip through the catheter, the blood rich in CTC is converged at the center of liquid flow through the converging and diverging unit, and finally the CTC is collected at the collecting port; the CTC-removed blood is split through the split channel to the recovery port of the microfluidic chip and is returned to the living animal/patient through the intravenous catheter.

The dialysis device/test system of fig. 12 can also be operated in another mode, in which living animal/patient blood flows into the microfluidic chip through the catheter, CTCs are concentrated to be collected at the collection port, and almost all CTCs in the blood circulation are removed by multiple cycle collection and separation; the separated and purified blood without CTCs is returned to the living animal/patient via an intravenous catheter.

Further, with the system for separating and qualitatively and quantitatively detecting tumor cells in systemic circulation shown in fig. 12, the whole dialysis process is automatically performed, blood enters the main inlet 1 of the microfluidic chip through the peristaltic pump, target particles CTC are concentrated and collected at the collecting port 4, and other blood cells (such as white blood cells, red blood cells and platelets) directly flow to the shunt port 5, and the blood sample feedback can be directly realized by connecting the shunt port 5 of the microfluidic chip 902 with the blood vessel of the living animal/patient.

In the dialysis device of the present invention, a single microfluidic chip comprising one convergent split cascade may be used, two or more convergent split cascades connected in series and/or in parallel may be included on one microfluidic chip as required, or a plurality of microfluidic chips comprising one or more convergent split cascades may be used. Wherein, the series connection can realize better enrichment and concentration effects, and the parallel connection can realize higher flux.

In the dialysis device/detection system of the present invention, the CTC enrichment and isolation module may further comprise a CTC staining imaging module comprising a microscope 903 and a PC computer 904. The CTC staining imaging module may stain, count, and/or identify the collected CTCs. Meanwhile, according to the test requirements of subsequent multiunit science, drug screening and the like, the collected CTC can be further analyzed.

The invention also provides a method for separating the in-vivo circulating tumor cells by adopting the system for separating and quantitatively detecting the in-vivo circulating tumor cells, wherein the blood of a living animal/patient is subjected to circulating enrichment, concentration and separation in a CTC enrichment and separation module, and the circulating tumor cells in the blood are separated, so that the qualitative and quantitative detection of CTC can be realized by methods of specific staining, multi-group chemical detection and the like, and real-time multi-group chemical full-spectrum personalized information is obtained; the blood extracorporeal circulation module is used for conveying other blood cells flowing out from the outlet of the microfluidic chip back to the living animal/patient. In some embodiments of the invention, isolated circulating tumor cells may be detected and counted using immunofluorescent staining methods using the nuclear dye Hoechst and fluorescent-labeled pan-CK, CD45 antibodies to stain and identify CTCs at the capture unit.

The blood of living animals/patients is circulated in a high-efficiency CTC enrichment and separation module for enrichment, concentration and separation, wherein the CTC enrichment and separation module can adopt a single design, and can realize serial-parallel connection on a chip according to the requirement. And placing the microfluidic chip on a microscope (03), and collecting a large number of CTCs through a chip collecting port (4) of the chip after a certain time, wherein dialysis is stopped. After which CTC fluorescent markers were introduced and after 30min of labelling the cells were visualized. The number of CTCs in the blood of the living animal/patient was counted.

The method for using the system for dialyzing and quantitatively detecting the tumor cells in the systemic circulation is as follows:

(1) Sterilizing with alcohol or ultraviolet irradiation to ensure sterility of all extracorporeal devices, soaking all the pipelines with heparin to prevent coagulation reaction, and pre-filling with physiological saline to prevent bubbles. Living animals/patients began instilling heparin half an hour prior to the experiment.

(2) The arterial or venous connection conduit 915 and the conduit 916 are connected to the carotid artery and the jugular vein of the living animal or to the median elbow artery and the median elbow vein of the patient respectively, the microfluidic chip is connected to the conduit 921 and the conduit 922, and the peristaltic pump 913 and the control valves 911 and 912 are sequentially opened to perform dialysis on the premise of ensuring tightness. As blood flows through the microfluidic chip 902, CTCs are enriched and concentrated and collected at the chip collection port 4, and other smaller blood cells are returned to the body through the chip shunt port 5. Because the flow resistance of the chip is smaller, the blood flow speed in the whole circulation process is proper, and therefore, the vital sign is not influenced by the dialysis.

(3) After a certain amount of time has elapsed, the peristaltic pump 913 and the control valves 911 and 912 are closed.

(4) The arterial or venous connection 915 and 916 catheters are removed from the animal/patient and the whole dialysis procedure is completed.

(5) The CTC collected by the chip collection port 4 is drained into a 96-well plate, and then nuclear dye Hoechst and fluorescent-labeled pan-CK and CD45 antibodies are added to dye and identify the CTC. After 30min of labelling, the cells were visualized. Furthermore, the number of CTCs in the blood of the living animal/patient can be counted.

Example 13

A BALB/c mouse model of breast cancer lung metastasis is established by tail vein injection of firefly luciferase labeled mouse breast cancer cells (4T 1-luc). Specific operation for 10 BALB/c Small100 mu L of rat tail intravenous injection with the density of 10 ⁶ Per mL of 4T1-luc cells, significant lung metastasis was observed by in vivo fluorescence imaging of the small animals on day 5. The vascular duct embedding model of the BALB/c mouse is established, and the specific scheme mainly comprises that the carotid artery and the jugular vein of the mouse are inserted into the duct and are closed, and the mouse can still eat and survive normally under the condition of neck cannula. Model maintenance mainly comprises opening a closed catheter, sucking original sealing liquid (50% glucose, containing 500IU/mL heparin), injecting a certain volume of new sealing liquid, and sterilizing the catheter with alcohol or other sterilizing liquid.

(1) All the external devices are sterilized by using a high-temperature high-pressure steam sterilizing pot, and are wiped by using alcohol before being used so as to ensure sterility. All the pipes are soaked by heparin to prevent coagulation reaction, and are pre-filled with physiological saline to prevent bubbles.

(2) As shown in fig. 12, an arterial or venous connection catheter is respectively connected to carotid artery and jugular vein of each mouse (BALB/c breast cancer lung metastasis model mice formed by inoculating 4T1 cancer cells of the same genus through tail vein and subcutaneous, a microfluidic chip (11 layers or 16 layers of microfluidic chips) is connected to the catheter, and a peristaltic pump and a control valve are sequentially opened to perform dialysis on the premise of ensuring tightness, wherein in the embodiment, the arterial or venous connection catheter has an inner diameter of 0.8mm and an outer diameter of 1.2mm; the inner diameter of the catheter is 0.5mm and the outer diameter is 1mm.

(3) After a period of time, the peristaltic pump and control valve are closed after the dialyzed blood reaches a certain volume.

(4) The arterial or venous connection catheter was removed from the mouse blood vessel and the whole dialysis procedure was completed.

(5) The CTC collected by the collection port is introduced with a fluorescent marker comprising a nuclear dye Hoechst and a pan-CK and CD45 antibody with fluorescent markers, and the cells are made visible after 30min of marking. Furthermore, the number of CTCs in the blood of the mice can be counted.

As shown in fig. 13, the result of CTC filtration is shown in fig. 13, and it can be seen that the design of the chip can achieve higher capture efficiency (the average value of capture efficiency is 95.7%, and the capture efficiency=the number of collected CTCs/(the number of collected ctcs+the number of non-collected CTCs)) in a larger flow rate range (the flow rate of in vivo experiments is about 2mL/h, because the whole blood of the mice is less, the blood flow is relatively slow, but the flow rate range in vitro experiments can reach 5-40 mL/h), so that better CTC separation is achieved.

The CTC enrichment and separation module of the present invention is not limited to the structure in the above embodiments, and may be a plurality of microfluidic chips connected in series-parallel or combined in any other manner. The number of the converging and diverging units of the microfluidic chip can be any value. The number of the convergence structures in the convergence and distribution unit can be any value.

The foregoing is merely a partial embodiment of the present invention and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims (16)

1. A microfluidic chip comprising a main inlet (1), a main channel (2), one or more converging and diverging units (3), a collecting port (4) and a diverging port (5) arranged in series, wherein the main inlet (1), the one or more converging and diverging units (3) and the collecting port (4) are connected by the main channel (2) and are in fluid communication;

wherein the chip is configured such that fluid enters the main channel (2) via the main inlet (1) and flows through the one or more converging and diverging units (3), each converging and diverging unit (3) converges target particles in the fluid at the center of the fluid flow, the fluid rich in target particles enters the latter converging and diverging unit or flows out via the collecting port (4), and the fluid with reduced content of target particles flows out from the diverging port (5).

2. The microfluidic chip according to claim 1, wherein each of the converging and diverging units (3) comprises a secondary inlet (31), one or more converging structures (32) arranged in series, and a diverging channel (33);

wherein the converging and diverging units are configured such that fluid flowing in from the main inlet (1) or the front converging and diverging unit enters the one or more converging structures (32) arranged in series via the secondary inlet (31), each converging structure (32) converges target particles in the fluid at the center of the fluid flow, and fluid rich in target particles enters the rear converging structure or the rear converging and diverging unit, or flows out via the collecting port (4), and fluid with reduced content of target particles enters the diverging port (5) via the diverging channel (33).

3. The microfluidic chip according to claim 2, wherein each of the converging structures (32) comprises a central channel (321) and a lateral tributary channel (322); wherein the central channel is connected with the main channel at both ends; the side branch passage is located at the side of the central passage and intersects the main passage and the central passage at both ends.

4. A microfluidic chip according to claim 2 or 3, wherein the shunt channels (33) are located downstream of the final concentration structure and distributed beside the main channel.

5. The microfluidic chip according to any one of claims 1 to 4, wherein the fluid with reduced target particle content in each converging flow splitting unit of the series arrangement enters the common flow splitting bus (34) via a respective flow splitting channel (33) and is released via a common flow splitting port (5), or

The fluid with reduced content of target particles in each converging and diverging unit of the series arrangement enters the respective diverging port (5) via the respective diverging channel (33) for release.

6. Microfluidic chip according to anyone of claims 1 to 5, further comprising a filter unit (6) located before the main inlet (1) and the first stage converging and diverging unit, such that fluid entering the main inlet flows through the filter unit before entering the converging and diverging unit.

7. The microfluidic chip according to any one of claims 1 to 6, which does not comprise a capture unit, but is configured such that the fluid enriched in target particles flows out of the final converging and diverging unit directly via the collection port (4).

8. The microfluidic chip according to any one of claims 1 to 7, wherein the one or more series arranged converging and diverging units are referred to as a converging and diverging unit cascade, the chip comprising 2 or more parallel arranged said converging and diverging unit cascades.

9. The microfluidic chip of claim 8, comprising a multilayer structure, wherein each layer of the multilayer structure comprises 1 or more of the converging split cell cascades arranged in parallel, and the converging split cell cascades of each layer are connected in parallel, in series, or both in parallel and in series.

10. A device for separating, enriching, concentrating, collecting, removing and/or analyzing target particles comprising a microfluidic chip according to any one of claims 1 to 9.

11. The device of claim 10, which is a dialysis device.

12. The device according to claim 10 or 11, further comprising a filtration unit (6) located upstream of and independent of the microfluidic chip.

13. A method of separating, enriching, concentrating, collecting, removing and/or analyzing target particles comprising:

(1) Providing a fluid containing or to be determined whether to contain target particles, and

(2) Flowing the fluid through a microfluidic chip according to any one of claims 1 to 9 or a device according to any one of claims 10 to 12.

14. The method of claim 13, which is a dialysis method comprising

(1) Providing a fluid to be dialyzed, wherein the fluid to be dialyzed comprises or is suspected to comprise target particles,

(2) Flowing the fluid through a microfluidic chip according to any one of claims 1 to 9 or a dialysis device according to claim 11 or 12, and

(3) The fluid from the shunt port with reduced target particle content is collected.

15. The method according to claim 13 or 14, further comprising

(4) The fluid enriched in target particles from the collection port is collected.

16. The microfluidic chip according to any one of claims 1 to 9, the device according to any one of claims 10 to 12, the method according to any one of claims 13 to 15, wherein the fluid is blood, plasma, serum, perfusion fluid, ascites, leukopenia enrichment fluid, urine, tissue fluid, cerebrospinal fluid, cell culture fluid or cell mixture fluid, preferably blood, perfusion fluid, ascites or leukopenia enrichment fluid, more preferably blood, and/or

The target particles are tumor cells, preferably circulating tumor cells.

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