Three-dimensional fluid focusing structure, hardware device and application suitable for microfluidic impedance cytometer

Abstract

The application provides a three-dimensional fluid focusing structure, a hardware device and application of the three-dimensional fluid focusing structure suitable for a microfluid impedance cytometer, and mainly comprises a specific structure, an implementation method and a supporting hardware system. In the structure, a sheath flow is an insulating solution, a sample flow is a high-conductivity solution, and cells pass through the sample flow and block electric field lines to realize impedance detection. The structure adopts a "sleeve" structure in which a sheath flow conduit wraps a sample flow conduit, focuses the sample flow on a central axis and realizes three-dimensional focusing. The sheath flow and the sample flow are quickly separated through a sample flow expansion channel of a disc-shaped separation structure, and the structure is suitable for a microfluid impedance cytometer. Compared with the existing method, the application adopts fluid focusing, avoids problems such as channel blockage, adjusts a flow rate ratio of the sheath flow and the sample flow to control a focusing size, detects cells of different sizes, utilizes a sample flow expansion channel to ensure the sensitivity of impedance detection, and therefore, high-sensitivity and high-throughput detection of single-cell impedance data can be realized.

Description

Technical Field

[0001] This invention relates to a three-dimensional fluid focusing structure, hardware device, and application suitable for microfluidic impedance cytometers, belonging to the field of cell detection technology. Background Technology

[0002] Blood is an essential component of the human body, responsible for transporting nutrients and metabolic waste, among other vital functions. Peripheral blood cells mainly consist of three categories: red blood cells, white blood cells, and platelets. White blood cells play an irreplaceable role in the human immune system and can be further divided into neutrophils, eosinophils, basophils, monocytes, and lymphocytes. The number and state of blood cells vary depending on a person's health status. For example, smaller red blood cells are commonly seen in iron deficiency anemia and anemia due to chronic disease, while sickle-shaped red blood cells are common in sickle cell anemia. Therefore, accurately determining the number and state of blood cells is helpful in understanding the body's condition, diagnosing diseases, and guiding medication use.

[0003] Currently, commercial hematology analyzers utilize the Coulter principle to count red blood cells, white blood cells, and platelets. The Coulter principle states that when a DC voltage is applied to electrodes inserted on either side of a small aperture, cells passing through the aperture block the electric field lines, causing impedance pulses. The number of impedance pulses represents the number of cells that have passed through the aperture, thus enabling cell counting. The amplitude of the impedance pulses is proportional to the cell size. The advantage of this method is that single cells flow through the detection area one by one, allowing for rapid cell counting. The disadvantage is that because cells occupy a relatively small volume in the detection area, the degree to which they block the electric field lines is limited, resulting in small changes in impedance pulses, meaning the sensitivity of impedance detection is relatively low.

[0004] Microfluidic impedance cytometers based on solid channels mainly include two methods: microchannel-based and compression-channel-based. The detection principle of microchannel-based microfluidic impedance cytometers is as follows: the cross-sectional area of ​​the microchannel is larger than the cell size. A pair of metal electrodes are integrated on the inner wall of the microchannel. An AC voltage is applied to the electrodes. When a cell passes through the electrode position, it blocks the electric field lines, causing a current change, which is then converted into an impedance pulse. The advantage of this method is its high detection throughput; the disadvantage is that the current change is affected by the distance between the cell and the electrodes, and the proportion of electric field lines blocked by the cell is small, resulting in only a weak current change, i.e., low impedance detection sensitivity. The detection principle of compression-channel-based microfluidic impedance cytometers is as follows: the cross-sectional area of ​​the compression channel is smaller than the cell size. A pair of metal electrodes are integrated on the bottom surface of the compression channel. An AC voltage is applied to the electrodes. After being squeezed, the cell passes through the compression channel, completely blocking the electric field lines, causing a larger impedance pulse. Furthermore, the electrical properties of the cell can be obtained based on an electrical model. The advantage of this method is its large current variation and high sensitivity to the electrical properties of cells, allowing for further characterization of the cells' biophysical and electrical properties, i.e., high impedance detection sensitivity. The disadvantage is that it is limited by the cross-sectional area of ​​the compression channel, which can only accommodate cells within a certain size range, and the compression channel is prone to clogging, resulting in relatively low detection throughput. In summary, current microfluidic impedance cytometers based on solid channels present a trade-off between detection sensitivity and detection throughput. When the channel size is larger than the cell size, the detection throughput is high while the impedance change caused by the cell is low; conversely, when the channel size is smaller than the cell size, the impedance detection sensitivity is high, but the channel is prone to clogging, leading to low detection throughput.

[0005] Fluid-focused microfluidic impedance cytometers can achieve small detection areas while avoiding channel blockage, mainly including two methods: two-dimensional focusing and three-dimensional focusing. Two-dimensional fluid focusing can be divided into focusing in the width or height direction of the microchannel. Taking a microfluidic impedance cytometer that achieves fluid focusing in the width direction as an example, its detection principle is as follows: A pair of metal electrodes are integrated on the channel wall of the microchannel and an AC voltage is applied. The conductive sample flow of suspended cells is focused within a certain dimension in the width direction by the insulating sheath flow, thereby restricting the distribution of electric field lines in the width direction. When the cell passes through the electrode position, it blocks most of the electric field lines in the width direction, while in the height direction, the cell only blocks part of the electric field lines. This method has a high detection throughput, but it can only improve the sensitivity of impedance detection to a certain extent. Three-dimensional fluid focusing simultaneously focuses the sample stream along both the width and height of a microchannel. The detection principle of a microfluidic impedance cytometer based on three-dimensional focusing is as follows: a pair of metal electrode wires are connected to the inlet and outlet of the microchannel and an AC voltage is applied. An insulating sheath current focuses the conductive sample stream onto the central axis of the channel, simultaneously restricting the distribution of electric field lines in both the width and height directions. As cells travel through the sample stream, they block most of the electric field lines, causing a significant impedance change. This method has high detection throughput; however, existing three-dimensional fluid focusing detection methods typically focus the sample stream to a small size, failing to achieve sample stream expansion, resulting in a high impedance baseline that masks the impedance changes caused by cell passage.

[0006] Therefore, it is of great significance to develop a three-dimensional fluid focusing structure suitable for microfluidic impedance cytometers. Summary of the Invention

[0007] This invention provides a three-dimensional fluid focusing structure, hardware device, and application suitable for microfluidic impedance cytometers. The three-dimensional fluid focusing structure for microfluidic impedance cytometers mainly includes a specific structure, an implementation method, and a hardware system for measuring cell impedance that matches the structure. This structure uses a non-conductive fluid as the sheath flow and a highly conductive fluid as the sample flow. Cells travel through the three-dimensionally focused sample flow and block electric field lines, thus achieving impedance detection of individual cells. To achieve three-dimensional fluid focusing, the structure uses a "sleeve" structure where the sheath flow conduit wraps around the sample flow conduit, focusing the sample flow onto the central axis of the channel. For suitability for microfluidic impedance cytometers, the structure utilizes a disk-shaped separation structure within the sample flow expansion channel to achieve rapid separation of the insulating sheath flow and the conductive sample flow.

[0008] This invention utilizes microfluidic technology to develop a three-dimensional fluid focusing structure suitable for microfluidic impedance cytometers. Compared to existing technologies, this method offers two main improvements:

[0009] (1) Among the existing microfluidic impedance cytometers based on solid microchannels, the impedance change caused by cells is relatively small in the detection methods based on microchannels; the detection methods based on compressed channels are limited by the size of the compressed channels, the compressed channels are easily blocked, the detection throughput is reduced, and they can only detect the impedance of single cells within a certain size range.

[0010] To address this drawback, this invention designs a three-dimensional fluid focusing channel, which can improve the detection throughput of single cells by using fluid focusing; and by adjusting the fluid flow rate to control the focusing size, it can detect the impedance of single cells in different size ranges.

[0011] (2) Among the existing microfluidic impedance cytometers based on fluid focusing, the detection method based on two-dimensional fluid focusing can only improve the sensitivity of impedance detection to a certain extent. The detection method based on three-dimensional fluid focusing is difficult to expand after the sample flow is focused to a smaller size, and the impedance changes caused by cells are masked by the higher impedance baseline.

[0012] To address this drawback, this invention designs a disk-shaped separation structure. Four disk-shaped separation structures are connected in series after the focusing channel and connected to the external atmospheric pressure. This achieves three-dimensional expansion of the sample flow before the outlet, ensuring high sensitivity of impedance detection.

[0013] The present invention adopts the following technical solution:

[0014] A three-dimensional fluid focusing structure suitable for microfluidic impedance cytometers includes a sample flow inlet channel, a sheath flow inlet channel, a three-dimensional focusing channel, a sample flow extension channel, a sheath flow outlet channel, and a sample flow outlet channel;

[0015] Sample flow inlet channel for introducing a conductive sample flow of suspended cells;

[0016] A sheath flow inlet channel is used to introduce a non-conductive fluid so that the non-conductive fluid completely encapsulates the sample flow;

[0017] The three-dimensional focusing channel is connected to the rear end of the sample flow inlet channel and the rear end of the sheath flow inlet channel. The three-dimensional focusing channel focuses the sample flow in cross-section onto the central axis of the three-dimensional focusing channel.

[0018] The sample flow expansion channel is connected to the rear end of the 3D focusing channel to help the focused sample flow expand in three dimensions;

[0019] The sheath flow outlet channel is connected to the sample flow expansion channel;

[0020] The sample stream export channel is connected to the back end of the sample stream extension channel.

[0021] Furthermore, the three-dimensional fluid focusing structure is formed by printing insulating resin material using 3D printing technology; optionally, the processing of the three-dimensional fluid focusing structure uses 3D printing technology and photosensitive resin insulating material, and the three-dimensional focusing structure is fabricated in an integrated manner based on photocuring method, and can realize the alignment of the sample flow inlet channel and the sheath flow inlet channel to ensure that the sample flow is focused on the central axis of the three-dimensional focusing channel.

[0022] Furthermore, the cross-section of the sample flow inlet channel is circular, and the diameter of the sample flow inlet channel gradually changes from about 1000 micrometers to about 100 micrometers.

[0023] Furthermore, the sheath flow inlet channel consists of two circular channels symmetrically distributed on both sides of the sample flow inlet channel to completely envelop the sample flow with the non-conductive fluid; preferably, the cross-sectional diameter of each circular channel of the sheath flow inlet channel is 500 micrometers.

[0024] Furthermore, the three-dimensional focusing channel is a circular channel, which focuses the sample flow with a circular cross-section onto the central axis of the three-dimensional focusing channel; optionally, the cross-sectional diameter of the three-dimensional focusing channel is 50 micrometers, and the diameter of the focused sample flow varies within the range of 0 to 50 micrometers; optionally, if the sample flow diameter is 0 micrometers, no sample flow is introduced; optionally, if the sample flow diameter is 50 micrometers, no sheath flow is introduced.

[0025] Furthermore, the sample flow expansion channel consists of four disk-shaped separation structures connected to the rear end of the three-dimensional focusing channel, which help to expand the focused sample flow in three dimensions (after passing through the first disk-shaped structure, the focused sample flow with a diameter of about 5 micrometers can be expanded to a diameter of about 10 micrometers); optionally, each disk-shaped separation structure has a diameter of 150 micrometers and a thickness of 50 micrometers.

[0026] Furthermore, the sheath flow outlet channel is a symmetrical rectangular and circular channel connected to the sample flow expansion channel on the left and right; preferably, the rectangular channel is 75 micrometers away from the central axis of the sample flow expansion channel, with a height of 200 micrometers and a width of 350 micrometers; preferably, the diameter of the circular channel gradually changes from 500 micrometers to 1000 micrometers.

[0027] Furthermore, the sample flow outlet channel is a circular channel connected to the rear end of the sample flow extension channel; preferably, the cross-sectional diameter of the sample flow outlet channel gradually changes from 500 micrometers to 1000 micrometers.

[0028] Further, the non-conductive fluid is an insulating sheath flow; optionally, the sheath flow is a deionized water insulating solution containing dissolved sucrose, a fluorinated oil insulating solution, or a mineral oil insulating solution.

[0029] Optionally, the sample stream is phosphate buffer, physiological saline, or a conductive solution of cell culture medium;

[0030] Optionally, the sample flow expansion channel in the three-dimensional fluid focusing structure includes multiple disk-shaped separation structures;

[0031] Optionally, the channel cross-section in the three-dimensional fluid focusing structure is circular, rectangular, or semi-circular.

[0032] A hardware device suitable for a microfluidic impedance cytometer, the hardware device comprising a three-dimensional fluid focusing structure, a fluid control module, an impedance measurement module, and a cell imaging module as described above;

[0033] The fluid control module includes a fluid injection pump and a liquid delivery hose; the fluid injection pump is connected to the three-dimensional fluid focusing structure via the liquid delivery hose; optionally, the fluid injection pump can be manually controlled to output the desired flow rate; optionally, for a syringe with a capacity of 1 mL, the fluid injection pump can provide an adjustable flow rate in the range of 0.0033 μL / min to 205.30 mL / hr.

[0034] The impedance measurement module is connected to the sample flow inlet channel and sample flow outlet channel of the three-dimensional fluid focusing structure by inserting a metal wire; optionally, the impedance measurement module can detect impedance with an amplitude of about 1MΩ at four frequencies, and the output frequency is 100,000 sampling points / second; optionally, the impedance measurement module includes a lock-in amplifier and a data acquisition card;

[0035] The cell imaging module includes an inverted microscope and a high-speed camera. The inverted microscope is used to observe and measure the sample flow size and cell movement process in the three-dimensional focusing channel (107). The high-speed camera can control the resolution of the recorded video to be in the range of 128*8-1920*1200, the frame rate to be in the range of 730-400000, and the minimum exposure time to be 1μs. It is combined with the inverted microscope to acquire images and videos of cells moving through the three-dimensional focusing channel.

[0036] The application of the hardware device described above in measuring cell impedance includes the following steps:

[0037] First, connect the three-dimensional fluid focusing structure, fluid control module, impedance measurement module, and cell imaging module. The connection method is as follows: the fluid output end of the fluid control module is connected to the sample flow inlet channel and the sheath flow inlet channel, respectively; the measurement end of the impedance measurement module is connected to the sample flow inlet channel and the sample flow outlet channel in the three-dimensional fluid focusing structure through the insertion electrode line, and AC voltages at four frequencies are applied to the insertion electrode line; the imaging optical path of the cell imaging module is placed at the three-dimensional focusing channel in the three-dimensional fluid focusing structure.

[0038] Then, a conductive sample stream of suspended cells is added to the fluid input end of the sample stream inlet channel, and an insulating sheath stream is added to the fluid input end of the sheath stream inlet channel. Sucrose is dissolved in the insulating sheath stream to maintain an osmotic pressure equal to that of the cells, so as to avoid cell rupture or shrinkage caused by osmotic pressure mismatch. The flow rates of the conductive sample stream and the insulating sheath stream are controlled by the fluid control module. The sample stream is three-dimensionally focused into a circle by the sheath stream, while more than 50% of the electric field generated by the AC voltage is confined in the sample stream. The size of the focused sample stream is controlled by adjusting the flow rate ratio of the sample stream to the sheath stream.

[0039] Next, the cells suspended in the sample stream are driven by the fluid to travel through the sample stream, passing sequentially through the three-dimensional focusing channel, the sample stream expansion channel, and the sample stream exit channel. The impedance measurement module is used to detect the impedance data when there are cells passing between the two inserted electrode lines. At the same time, the cell imaging module is used to collect image and video data during the cell's passage. The impedance data and image and video data are used as the raw data for the experiment.

[0040] The beneficial effects of this invention are:

[0041] As can be seen from the above description of the technical solution, the present invention, a three-dimensional fluid focusing structure suitable for microfluidic impedance cytometers, has the following beneficial effects:

[0042] (1) This invention expands the detection size range of single-cell impedance. Compared with existing methods, the size of the suspended cell sample stream is controllable, thus matching cells of different sizes.

[0043] (2) This invention improves the detection throughput of single-cell impedance. Compared with existing methods, the fluid focusing method effectively avoids channel blockage, thus achieving a higher detection throughput. Attached Figure Description

[0044] Figure 1 A schematic diagram of the hardware device applicable to a microfluidic impedance cell analyzer according to the present invention;

[0045] Figure 2 Schematic diagram of the three-dimensional fluid focusing module: I. Overall structure schematic diagram; II. Top view of key structure; III. Side view of sheath flow outlet channel.

[0046] Figure 3 Physical image of the 3D fluid focusing module, I. Physical image, II. Enlarged view of a part;

[0047] Figure 4 Three-dimensional fluid focusing and expansion at different flow rates: Sample I: 5 μL / min; Sheath flow: 50 μL / min; Sample II: 5 μL / min; Sheath flow: 150 μL / min;

[0048] Figure 5Impedance signals during cell passage: Ia cell passage process; Ib cell passage process; Ic cell passage process; II impedance pulse;

[0049] In the figure, 1-3D fluid focusing module, 101-sheath flow inlet, 102-sample flow inlet, 103-sheath flow outlet, 104-sample flow outlet, 105-sheath flow introduction channel, 106-sample flow introduction channel, 107-3D focusing channel, 108-sheath flow export channel, 109-sample flow expansion channel, 110-sample flow export channel; 2-fluid control module; 3-impedance measurement module; 4-cell imaging module. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the scope of protection of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0051] This embodiment mainly consists of hardware devices and experimental procedures.

[0052] Figure 1 This is a schematic diagram of the hardware device for a microfluidic impedance cytometer according to the present invention. The hardware device is a device required for measuring cell impedance, matching the three-dimensional fluid focusing structure of the microfluidic impedance cytometer of the present invention. The three-dimensional fluid focusing structure is a three-dimensional fluid focusing module 1. The hardware device required in this embodiment is as follows: Figure 1 As shown, it mainly consists of four parts: a three-dimensional fluid focusing module 1, a fluid control module 2, an impedance measurement module 3, and a cell imaging module 4.

[0053] Figure 2 This is a schematic diagram of the 3D fluid focusing module. The 3D fluid focusing module 1 is the core module of the hardware device, formed by 3D printing of insulating resin material. The overall structure is shown in the schematic diagram below. Figure 2 As shown in Figure I. The three-dimensional fluid focusing module 1 sequentially includes a sample flow inlet channel 106, a sheath flow inlet channel 105, a three-dimensional focusing channel 107, a sample flow expansion channel 109, a sheath flow outlet channel 108, and a sample flow outlet channel 110, as shown in Figure I. Figure 2Figure II shows a top view of the key structure. A sample flow inlet 102 is provided at the end of the sample flow inlet channel 106. A sheath flow inlet 101 is provided at the end of the sheath flow inlet channel 105. Sheath flow outlets 103 are provided at both ends of the sheath flow outlet channel 108. A sample flow outlet 104 is provided at the end of the sample flow outlet channel 110. Specifically, the sample flow inlet channel 106 introduces a high-conductivity sample flow of suspended cells. Its structural feature is a circular cross-section, with a cross-sectional area much larger than the cell size to ensure smooth cell flow. The diameter of this channel's cross-section gradually decreases from approximately 1000 micrometers to approximately 100 micrometers (most cells have a diameter of approximately 15-20 micrometers). The sheath flow inlet channel 105 introduces a non-conductive fluid. Its structural feature is two circular channels symmetrically distributed on both sides of the sample flow inlet channel 106 to completely enclose the sample flow. The cross-sectional diameter of each circular channel in the sheath flow inlet channel 105 is approximately 500 micrometers. The three-dimensional focusing channel 107 is used to focus the sample flow. Its structural feature is a circular channel connected to the rear ends of the sample flow inlet channel 106 and the sheath flow inlet channel 105, focusing the sample flow with a circular cross-section onto the central axis of the three-dimensional focusing channel 107. The cross-sectional diameter of the three-dimensional focusing channel 107 is approximately 50 micrometers. The focused sample... The diameter of the sample flow can vary from 0 to 50 micrometers (if the sample flow diameter is 0 micrometers, no sample flow is introduced; if the sample flow diameter is 50 micrometers, no sheath flow is introduced); the structural feature of the sample flow expansion channel 109 is four disk-shaped separation structures connected to the rear end of the three-dimensional focusing channel 107, which help the focused sample flow to expand in three dimensions (after passing through the first disk-shaped structure, the focused sample flow with a diameter of about 5 micrometers can be expanded to a diameter of about 10 micrometers). The diameter of the disk-shaped separation structure is about 150 micrometers and the thickness is about 50 micrometers; the structural feature of the sheath flow outlet channel 108 is a symmetrical rectangular and circular channel connected to the sample flow expansion channel 109 on both sides. The rectangular channel makes the internal pressure of the sample flow expansion channel as equal as possible, about 75 micrometers from the central axis of the sample flow expansion channel 109, with a height and width of about 200 micrometers and 350 micrometers, respectively. The diameter of the circular channel gradually changes from about 500 micrometers to about 1000 micrometers to quickly discharge the sheath flow, as shown in the attached figure. Figure 2 Figure III shows a side view of the sheath flow outlet channel; the structural feature of the sample flow outlet channel 110 is a circular channel connected to the rear end of the sample flow extension channel 109, with a cross-sectional area much larger than the cell size to ensure smooth cell outflow, and the diameter of the cross-section gradually changes from about 500 micrometers to about 1000 micrometers.

[0054] The three-dimensional fluid focusing module 1 is fabricated using 3D printing technology and photosensitive resin insulating material. Based on a photopolymerization method, the three-dimensional focusing structure can be fabricated in one integrated unit, achieving high-precision alignment between the sample flow inlet channel 106 and the sheath flow inlet channel 105 to ensure the sample flow is focused on the central axis of the three-dimensional focusing channel 107, as shown in the attached figure. Figure 3As shown, attached Figure 3 The "I" in the image represents the actual product. Figure 3 II in the image is a magnified view of a key part.

[0055] The fluid control module 2 is a known technology and includes a fluid injection pump and a delivery tubing. The fluid injection pump can be manually controlled to output the desired flow rate. For a 1 mL syringe, the fluid injection pump can provide an adjustable flow rate in the range of 0.0033 μL / min to 205.30 mL / hr, and is connected to the aforementioned three-dimensional fluid focusing module 1 via the delivery tubing.

[0056] Impedance measurement module 3 is a known technology and includes a lock-in amplifier and a data acquisition card. According to the embodiment, it can detect impedance with an amplitude of approximately 1 MΩ at four frequencies, with an output frequency of 100,000 sampling points / second, and is connected to the sample flow inlet channel 106 and sample flow outlet channel 110 of the aforementioned three-dimensional fluid focusing module 1 via an inserted metal wire.

[0057] Cell imaging module 4 is a known technology, comprising an inverted microscope and a high-speed camera. The inverted microscope is used to observe and measure the sample flow size and cell movement process in the three-dimensional focusing channel 107. The high-speed camera can control the resolution of the recorded video within the range of 128*8-1920*1200, the frame rate within the range of 730-400000, and the minimum exposure time to 1μs. It is combined with the inverted microscope to acquire images and videos of cells moving through the three-dimensional focusing channel 107.

[0058] The specific experimental procedure in this embodiment is as follows:

[0059] First, connect the three-dimensional fluid focusing module 1, fluid control module 2, impedance measurement module 3, and cell imaging module 4. The connection method is as follows: the fluid output terminal of fluid control module 2 is connected to sample flow inlet channel 106 and sheath flow inlet channel 105, respectively; the measurement terminal of impedance measurement module 3 is connected to sample flow inlet channel 106 and sample flow outlet channel 110 in three-dimensional fluid focusing module 1 via inserted electrode lines, and AC voltages at four frequencies are applied to the inserted electrode lines; the imaging optical path of cell imaging module 4 is placed at the three-dimensional focusing channel 107 in three-dimensional fluid focusing module 1.

[0060] Then, a high-conductivity sample stream is added to the fluid input end of the sample stream inlet channel 106, and cells of a certain concentration are suspended in it. An insulating sheath stream is added to the fluid input end of the sheath stream inlet channel 105. Sucrose is dissolved in the insulating sheath stream to maintain an osmotic pressure equal to that of the cells, thus avoiding cell rupture or shrinkage caused by osmotic pressure mismatch. The flow rates of the conductive sample stream and the insulating sheath stream are controlled by the fluid control module 2. The sample stream is three-dimensionally focused into a circle by the sheath stream, while most of the electric field generated by the AC voltage is confined within the sample stream. The size of the focused sample stream is controlled by adjusting the ratio of the sample stream to the sheath stream flow rates, as shown in the attached figure. Figure 4 As shown. In the appendix Figure 4 In the diagram, the dark-colored solution in the middle is a phosphate buffer solution doped with ink, serving as the conductive sample stream; the insulating sheath stream utilizes a deionized aqueous solution of dissolved sucrose. When the sample flow rate is 5 μL / min and the sheath flow rate is 50 μL / min, the sample stream is focused to approximately 12 micrometers wide at the center of the three-dimensional focusing channel 107, and the sample stream is expanded at the end of the first sample stream expansion channel, as shown in the attached diagram. Figure 4 As shown in Figure I; when the sample flow rate is 5 μL / min and the sheath flow rate is 150 μL / min, the sample flow is focused to a width of approximately 7 μm, and the sample flow is expanded at the beginning of the third sample flow expansion channel, as shown in the attached figure. Figure 4 As shown in II in the diagram.

[0061] Next, the cells suspended in the sample stream are propelled through the sample stream by the fluid, passing sequentially through the three-dimensional focusing channel 107, the sample stream expansion channel 109, and the sample stream exit channel 110, as shown in the attached diagram. Figure 5 As shown in Figure I; impedance data is obtained by using impedance measurement module 3 to detect whether there are cells passing between the two inserted electrode lines, as shown in the attached figure. Figure 5 As shown in II; simultaneously, image and video data of the cell transit process are acquired using cell imaging module 4, and impedance data and image and video data are used as the raw data of the experiment.

[0062] This concludes the detailed description of this embodiment in conjunction with the accompanying drawings. Based on the above description, those skilled in the art should have a clear understanding of the three-dimensional fluid focusing structure applicable to microfluidic impedance cytometers of the present invention.

[0063] In this embodiment, the sheath flow is an insulating solution of deionized water containing dissolved sucrose, and the use of insulating solutions such as fluorinated oil and mineral oil does not affect the basic function; the sample flow is phosphate-buffered saline (PBS), and the use of conductive solutions such as physiological saline and cell culture medium does not affect the basic function.

[0064] In this embodiment, the three-dimensional fluid focusing module is formed by 3D printing using photosensitive resin material. However, the above-mentioned three-dimensional fluid focusing module can also be formed using different 3D printers and printing materials, as well as femtosecond laser processing technology.

[0065] In this embodiment, the sample flow expansion channel 109 in the three-dimensional fluid focusing module includes four disk-shaped separation structures, which can also be changed to different numbers without affecting the implementation of the basic functions.

[0066] The three-dimensional fluid focusing demonstrated in this invention is the basic unit of this method, which can easily arrange cells in parallel and series along the direction of cell travel, and even merge certain structures, which will bring different effects.

[0067] In this invention, the channel cross-section of the three-dimensional fluid focusing module 1 is circular, but it can also be replaced with a rectangular or semi-circular shape without affecting the implementation of the basic functions.

[0068] In this invention, fluid is used to drive the cell solution through the channel, but other methods can also be used, such as applying negative pressure to the sheath flow outlet channel and the sample flow outlet channel.

[0069] The parts of this invention not described in detail are well-known to those skilled in the art. The embodiments described above are merely preferred embodiments of the invention, and do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Various modifications and improvements to the technical solutions of this invention made by those skilled in the art without departing from the spirit of the invention should fall within the protection scope defined by the claims of this invention.

Claims

1. A three-dimensional fluid focusing structure suitable for microfluidic impedance cytometers, characterized in that, It includes a sample flow inlet channel (106), a sheath flow inlet channel (105), a three-dimensional focusing channel (107), a sample flow expansion channel (109), a sheath flow outlet channel (108), and a sample flow outlet channel (110). The sample flow inlet channel (106) is used to introduce a conductive sample flow of suspended cells; Sheath flow inlet channel (105) is used to introduce a non-conductive fluid so that the non-conductive fluid completely encapsulates the sample flow; The three-dimensional focusing channel (107) is connected to the rear end of the sample flow inlet channel (106) and the rear end of the sheath flow inlet channel (105). The three-dimensional focusing channel (107) focuses the sample flow with a circular cross-section on the central axis of the three-dimensional focusing channel (107). The sample flow expansion channel (109) is connected to the rear end of the three-dimensional focusing channel (107) to help the focused sample flow to expand in three dimensions; The sheath flow outlet channel (108) is connected to the sample flow expansion channel (109). The sheath flow outlet channel (108) is a symmetrical rectangular and circular channel that is connected to the sample flow expansion channel (109) on the left and right. The sample flow export channel (110) is connected to the rear end of the sample flow expansion channel (109); The sample flow extension channel (109) consists of four disk-shaped separation structures connected to the rear end of the three-dimensional focusing channel (107). The four disk-shaped separation structures are connected in series after the focusing channel and connected to the external atmospheric pressure. The three-dimensional extension of the sample flow is realized before the outlet, which can ensure the high sensitivity of impedance detection.

2. The structure according to claim 1, characterized in that, The three-dimensional fluid focusing structure is formed by printing insulating resin material using 3D printing technology. The processing of the three-dimensional fluid focusing structure uses 3D printing technology and photosensitive resin insulating material. The three-dimensional focusing structure is fabricated in an integrated manner based on photocuring method. It can also achieve the alignment of the sample flow inlet channel (106) and the sheath flow inlet channel (105) to ensure that the sample flow is focused on the central axis of the three-dimensional focusing channel (107).

3. The structure according to claim 1, characterized in that, The cross-section of the sample flow inlet channel (106) is circular, and the diameter of the cross-section of the sample flow inlet channel (106) gradually changes from 1000 micrometers to 100 micrometers.

4. The structure according to claim 1, characterized in that, The sheath flow inlet channel (105) consists of two circular channels symmetrically distributed on both sides of the sample flow inlet channel (106) to completely envelop the sample flow with the non-conductive fluid; each circular channel of the sheath flow inlet channel (105) has a cross-sectional diameter of 500 micrometers.

5. The structure according to claim 1, characterized in that, The three-dimensional focusing channel (107) is a circular channel that focuses the sample flow with a circular cross-section onto the central axis of the three-dimensional focusing channel (107). The cross-sectional diameter of the three-dimensional focusing channel (107) is 50 micrometers, and the diameter of the focused sample flow varies from 0 to 50 micrometers. If the sample flow diameter is 0 micrometers, no sample flow is introduced. If the sample flow diameter is 50 micrometers, then no sheath flow will be introduced.

6. The structure according to claim 1, characterized in that, Each disc-shaped separation structure has a diameter of 150 micrometers and a thickness of 50 micrometers.

7. The structure according to claim 1, characterized in that, The rectangular channel is 75 micrometers from the central axis of the sample flow extension channel (109), with a height of 200 micrometers and a width of 350 micrometers; the diameter of the circular channel gradually changes from 500 micrometers to 1000 micrometers.

8. The structure according to claim 1, characterized in that, The sample flow outlet channel (110) is a circular channel connected to the rear end of the sample flow extension channel (109); the cross-sectional diameter of the sample flow outlet channel (110) gradually changes from 500 micrometers to 1000 micrometers.

9. The structure according to claim 1, characterized in that, The non-conductive fluid is an insulating sheath flow; the sheath flow is a deionized water insulating solution containing dissolved sucrose, a fluorinated oil insulating solution, or a mineral oil insulating solution. The sample stream is a phosphate buffer, physiological saline, or conductive solution of cell culture medium. The sample flow expansion channel (109) in the three-dimensional fluid focusing structure includes multiple disk-shaped separation structures; The channel cross-section in the three-dimensional fluid focusing structure is circular, rectangular, or semi-circular.

10. A hardware device suitable for a microfluidic impedance cytometer, characterized in that, The hardware device includes the three-dimensional fluid focusing structure as described in claim 1, the fluid control module (2), the impedance measurement module (3), and the cell imaging module (4). The fluid control module (2) includes a fluid injection pump and a liquid guiding hose; the fluid injection pump is connected to the three-dimensional fluid focusing structure through the liquid guiding hose; the fluid injection pump can output the required flow rate by manual control; For a 1 mL syringe, the fluid injection pump can provide an adjustable flow rate in the range of 0.0033 µL / min to 205.30 mL / hr; The impedance measurement module (3) connects the sample flow inlet channel (106) and sample flow outlet channel (110) of the three-dimensional fluid focusing structure by inserting a metal wire; the impedance measurement module (3) can detect impedance with an amplitude of 1 MΩ at four frequencies and output a frequency of 100,000 sampling points / second; The impedance measurement module (3) includes a lock-in amplifier and a data acquisition card; The cell imaging module (4) includes an inverted microscope and a high-speed camera; the inverted microscope is used to observe and measure the sample flow size and cell transit process in the three-dimensional focusing channel (107), and the high-speed camera can control the resolution of the recorded video to 128. 8-1920 Within a range of 1200 frames per second, with a frame rate of 730-400000 frames per second and a minimum exposure time of 1 µs, it was combined with an inverted microscope to acquire images and videos of cells moving through a three-dimensional focusing channel (107).

11. The application of the hardware device of claim 10 in measuring cell impedance, characterized in that, Includes the following steps: First, connect the three-dimensional fluid focusing structure, the fluid control module (2), the impedance measurement module (3), and the cell imaging module (4). The connection method is that the fluid output end of the fluid control module (2) is connected to the sample flow inlet channel (106) and the sheath flow inlet channel (105) respectively. The measurement end of the impedance measurement module (3) is connected to the sample flow inlet channel (106) and the sample flow outlet channel (110) in the three-dimensional fluid focusing structure through the insertion electrode line, and AC voltages at four frequencies are applied to the insertion electrode line. The imaging optical path of the cell imaging module (4) is placed at the three-dimensional focusing channel (107) in the three-dimensional fluid focusing structure. Then, a conductive sample stream of suspended cells is added to the fluid input end of the sample stream inlet channel (106), and an insulating sheath stream is added to the fluid input end of the sheath stream inlet channel (105). Sucrose is dissolved in the insulating sheath stream to maintain an osmotic pressure equal to that of the cells, so as to avoid cell bursting or shrinkage caused by osmotic pressure mismatch. The flow rates of the conductive sample stream and the insulating sheath stream are controlled by the fluid control module (2). The sample stream is three-dimensionally focused into a circle by the sheath stream, while more than 50% of the electric field generated by the AC voltage is confined in the sample stream. The size of the focused sample stream is controlled by adjusting the flow rate ratio of the sample stream to the sheath stream. Next, the cells suspended in the sample stream travel through the sample stream under the drive of the fluid, and pass through the three-dimensional focusing channel (107), the sample stream expansion channel (109) and the sample stream exit channel (110) in sequence; the impedance measurement module (3) is used to detect the impedance data when there are cells passing between the two inserted electrode lines; at the same time, the cell imaging module (4) is used to collect image and video data during the cell travel process, and the impedance data and image and video data are used as the raw data of the experiment.

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