Micro-fluidic chip, blood separation device and blood separation method

Abstract

The invention relates to the technical field of biomedicine, in particular to a micro-fluidic chip, a blood separation device and a blood separation method. The micro-fluidic chip comprises a chip base body, a micro-channel, a micro-channel inlet and a micro-channel outlet, the micro-channel, the micro-channel inlet and the micro-channel outlet are arranged in the chip base body, a plurality of grooves are formed in the bottom wall of the micro-channel, and the micro-channel is distributed in an Archimedes spiral shape. The micro-fluidic chip provided by the invention comprises micro-channels distributed in an Archimedes spiral shape, a plurality of grooves are formed in the bottom walls of the micro-channels, sound wave processing is coupled to construct an oscillating micro-bubble array during blood separation, micro-bubble resonance is excited to realize efficient control on cells in blood, red blood cells, white blood cells and blood platelets are effectively separated, and the blood separation efficiency is improved. The efficient preparation of the high-purity plasma matrix is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a microfluidic chip, a blood separation device and a blood separation method. Background Art

[0002] Because plasma matrix is ​​rich in growth factors, it has important application value in the fields of orthopedic tissue repair, chronic wound treatment, and medical cosmetology. Currently, the preparation of clinical plasma matrix mainly relies on centrifugation, but this technology has significant defects. First, the centrifugation process is cumbersome and there are large individual differences among patients, which affects the final separation effect. At the same time, centrifugation causes up to 20-30% of platelets to be activated or ruptured, affecting the treatment effect. Secondly, for some patients with high blood viscosity, centrifugation cannot completely and effectively separate red blood cells during the preparation of their plasma matrix, limiting its application in special clinical scenarios. In addition, the traditional centrifugation scheme has a weak ability to control the final platelet concentration and cannot achieve the preparation of high-concentration platelet plasma matrix.

[0003] To overcome the shortcomings of centrifugation, microfluidics has been introduced for plasma matrix preparation. One study proposed a spiral channel separation method based on inertial focusing, using Dean vortices to separate red blood cells and platelets. However, its processing efficiency is limited by laminar flow stability, and separation purity drops sharply when the hematocrit exceeds 5%. Other studies have used membrane filtration combined with centrifugation, but the microporous structure is easily clogged by fibrin or aggregated red blood cells, resulting in a lifespan of less than 10 cycles when processing whole blood samples. In recent years, acoustofluidics has garnered widespread attention due to its non-contact and low-shear properties. Some researchers have published a method for separating blood cells using a standing wave acoustic field. However, traditional acoustic wave devices have difficulty distinguishing between similarly sized cell subpopulations (such as red blood cells and platelets) due to their long acoustic wavelengths. Furthermore, the acoustic radiation force decays sharply with decreasing cell size, resulting in inefficient manipulation of small particle components. While surface acoustic wave technology can localize the acoustic field using interdigitated electrodes, its energy is severely attenuated along the propagation direction, making it inefficient for processing high-concentration whole blood (acoustic penetration significantly decreases when the hematocrit exceeds 40%).

[0004] Therefore, providing a new blood separation method and blood separation device is of great significance for improving later clinical applications. Summary of the Invention

[0005] To address the above technical issues, the present invention provides a microfluidic chip, a blood separation device, and a blood separation method. The microfluidic chip comprises microchannels arranged in an Archimedean spiral pattern, with multiple grooves disposed on the bottom wall of the microchannels. During blood separation, the chip couples acoustic wave processing to create an oscillating microbubble array, which stimulates microbubble resonance to achieve efficient manipulation of blood cells, effectively separating red blood cells, white blood cells, and platelets, and efficiently preparing a plasma matrix.

[0006] In a first aspect, the present invention provides a microfluidic chip, comprising a chip substrate and a microchannel arranged inside the chip substrate, as well as a microchannel inlet and a microchannel outlet, wherein a plurality of grooves are arranged on the bottom wall of the microchannel, and the microchannel is distributed in the shape of an Archimedean spiral.

[0007] As a preferred technical solution of the present invention, the diameter of the groove is 10-100 μm, for example, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, etc.

[0008] As a preferred technical solution of the present invention, the distance between adjacent grooves is 10-500 μm, for example, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, etc.

[0009] The present invention regulates the size and spacing of blood microbubbles by adjusting the size and spacing of micropores, so that the resonance frequency thereof matches the external sound wave, thereby stimulating a localized strong sound field.

[0010] As a preferred technical solution of the present invention, the cross-sectional shape of the microchannel is rectangular, the width of the microchannel is 1-10 mm, for example, 1 mm, 2 mm, 5 mm, 8 mm, 10 mm, etc.; the height of the microchannel is 1-10 mm, for example, 1 mm, 2 mm, 5 mm, 8 mm, 10 mm, etc.

[0011] As a preferred technical solution of the present invention, the lead of the Archimedean spiral is 1-5 times the width of the microchannel, for example, it can be 1 time, 2 times, 3 times, 4 times or 5 times.

[0012] The present invention does not impose excessive restrictions on the length of the microchannel, which can be determined based on the separation efficiency of the expected product.

[0013] As a preferred technical solution of the present invention, the chip substrate is made of polydimethylsiloxane-modified glass.

[0014] As a preferred technical solution of the present invention, the surface of the microchannel is treated with oxygen plasma to improve its cleaning effect.

[0015] In a second aspect, the present invention provides a blood separation device, comprising the microfluidic chip described in the first aspect, as well as a piezoelectric transducer, an optical detection module, a temperature control module, and a system control module;

[0016] The piezoelectric transducer is used to provide sound waves to the microfluidic chip;

[0017] The optical detection module is used to monitor the concentration and separation effect of red blood cells and platelets in the microchannel of the microfluidic chip in real time;

[0018] The temperature control module is used to control the temperature inside the microchannel of the microfluidic chip;

[0019] The system control module is used to analyze the signals output by the optical detection module and the temperature control module, and control the piezoelectric transducer to dynamically adjust the sound wave frequency and boost amplitude, and control the temperature control module to dynamically adjust the temperature.

[0020] The blood separation device provided by the present invention integrates a microfluidic chip and a piezoelectric transducer, and constructs a microbubble resonance array during the blood separation process. The microbubble resonance efficiently manipulates red blood cells and white blood cells, causing them to adsorb on the microbubble surface in the groove to obtain a high-purity plasma matrix. Moreover, the microfluidic chip can be rinsed and reused after the blood separation is completed, effectively reducing costs. At the same time, the blood separation device provided by the present invention also integrates an optical detection module, a temperature control module and a system control module. The system control module identifies and analyzes the signal output by the optical detection module. When an abnormal signal is detected, it can control the piezoelectric transducer in real time to adjust the sound wave frequency or boost amplitude or control the temperature control module to adjust the temperature.

[0021] As a preferred technical solution of the present invention, the optical detection module includes a dual-wavelength absorbance sensor, which is installed throughout the microchannel of the microfluidic chip and is used to monitor the concentration and separation effect of red blood cells and platelets in the microchannel of the microfluidic chip in real time. The dual wavelengths are 405nm and 650nm, where the 650nm wavelength primarily detects the absorbance of red blood cells, and the 405nm wavelength primarily detects the absorbance of platelet-rich plasma matrix. The detection results can be used to determine the effectiveness of red blood cell removal at different locations.

[0022] In a third aspect, the present invention provides a blood separation method, which is performed using the blood separation device described in the second aspect, comprising:

[0023] Starting the blood separation device, setting the temperature of the temperature control module, injecting blood into the microchannel from the microchannel inlet of the microfluidic chip, setting the piezoelectric transducer to output an acoustic field to remove red blood cells and white blood cells, and obtaining a plasma matrix from the microchannel outlet, wherein the plasma matrix is ​​rich in platelets;

[0024] At the same time, the optical detection module monitors the concentration and separation effect of red blood cells and platelets in the microchannel of the microfluidic chip in real time. The system control module dynamically analyzes the signals output by the optical detection module and the temperature control module. When an abnormal signal is detected, the piezoelectric transducer is controlled to dynamically adjust the sound wave frequency and boost amplitude, or the temperature control module is controlled to dynamically adjust the temperature.

[0025] The blood separation method provided by the present invention has significant advantages over the existing technology:

[0026] (1) Microbubble resonance array design: By applying sound waves, a periodically oscillating microbubble array is constructed in the microfluidic chip. Under the action of sound waves, the blood microbubbles in the grooves are stimulated to resonate, thereby achieving the separation of cells of different sizes.

[0027] (2) Multi-stage dynamic separation mechanism: Combining the acoustic field with the spiral flow channel design, the differences in acoustic properties of red blood cells, white blood cells, platelets and other components in the plasma are utilized to achieve efficient separation of the plasma matrix with the help of enhanced acoustic radiation force and three-dimensional acoustic flow effect.

[0028] (3) Intelligent feedback system: Integrates optical sensing (optical detection module) and artificial intelligence algorithms (system control module) to achieve real-time adaptive control of sound field parameters; can adapt to samples of different blood types and pathological conditions (such as anemia or polycythemia), providing a technical basis for personalized medicine.

[0029] (4) Improved biocompatibility: Utilize temperature control modules and system control modules to achieve precise temperature control, ensuring cell activity and long-term operational stability.

[0030] Compared with traditional centrifugation methods, the method of the present invention provides an efficient and low-damage solution for bedside instant plasma matrix preparation, and is suitable for orthopedic repair, cosmetic medicine and other fields.

[0031] The acoustic field of the present invention uses high-frequency sound waves to excite the resonance of red blood cells and white blood cells in the micropores, accurately matching the acoustic resonance characteristics of red blood cells and white blood cells, capturing red blood cells and white blood cells in the grooves, so that the residual amount of red blood cells and white blood cells in the final plasma matrix is ​​less than 0.1%.

[0032] As a preferred technical solution of the present invention, the flow rate of the blood is 10-500 μL / min, for example, 10 μL / min, 20 μL / min, 30 μL / min, 40 μL / min, 50 μL / min, 100 μL / min, 200 μL / min, 500 μL / min, etc.

[0033] As a preferred technical solution of the present invention, the temperature control module is set to a temperature of 4-37°C, such as 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, etc.

[0034] As a preferred technical solution of the present invention, when a signal abnormality is detected, the piezoelectric transducer is controlled to dynamically adjust the sound wave frequency and the boost amplitude, or the temperature control module is controlled to dynamically adjust the temperature, specifically:

[0035] When it is detected that the red blood cell migration rate is higher than the set range, the piezoelectric transducer is controlled to increase the sound wave frequency, and the adjustment range of the sound wave frequency is 0-20%, such as 1%, 5%, 10%, 15%, 20%, etc.

[0036] When it is detected that the temperature exceeds the set range, the temperature control module is controlled to increase or decrease the temperature.

[0037] The technical solution provided by the embodiment of the present invention has the following advantages compared with the existing technology:

[0038] The microfluidic chip provided by the present invention includes microchannels distributed in the shape of Archimedean spirals, and multiple grooves are provided on the bottom wall of the microchannels. During blood separation, acoustic wave processing is coupled to construct an oscillating microbubble array, which stimulates microbubble resonance to achieve efficient manipulation of cells in the blood, effectively separate unexpected cells such as red blood cells and white blood cells, and realize efficient preparation of high-purity plasma matrix. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0041] Figure 1 Schematic diagram of the microfluidic chip according to Example 1 of the present invention.

[0042] Figure 2 This is a cross-sectional view of the microfluidic chip described in Example 1 of the present invention along the length direction of the microchannel. DETAILED DESCRIPTION

[0043] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features therein can be combined with each other.

[0044] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all the embodiments.

[0045] Example 1

[0046] This embodiment provides a microfluidic chip, and its planar schematic diagram is as follows Figure 1 As shown, the cross-sectional view along the length direction of the microchannel is as follows Figure 2 The chip substrate comprises a microchannel disposed within the chip substrate, as well as a microchannel inlet and outlet. The bottom wall of the microchannel is provided with multiple grooves arranged in an Archimedean spiral pattern. The diameter d of the grooves is 50 μm, and the distance l between adjacent grooves is 100 μm. The microchannel has a rectangular cross-section, a width D of 3 mm, a height h of 5 mm, a total length of 80 cm, and a lead L of the Archimedean spiral of 6 mm. The chip substrate is made of polydimethylsiloxane-modified glass, and the surfaces of the microchannels and grooves are treated with oxygen plasma.

[0047] Example 2

[0048] This embodiment provides a blood separation device with a size of less than 30×20×15cm. 3 , weighing less than 5 kg, comprising the microfluidic chip provided in Example 1 as well as a piezoelectric transducer, an optical detection module, a temperature control module, and a system control module;

[0049] Piezoelectric transducers are used to provide acoustic waves to the microfluidic chip;

[0050] The optical detection module includes a dual-wavelength absorbance sensor, which is set in the entire microchannel of the microfluidic chip. The optical detection module is used to monitor the concentration and separation effect of red blood cells and platelets in the microchannel of the microfluidic chip in real time;

[0051] The temperature control module is used to control the temperature inside the microchannel of the microfluidic chip;

[0052] The system control module is used to analyze the signals output by the optical detection module and the temperature control module, and control the piezoelectric transducer to dynamically adjust the sound wave frequency and boost amplitude, as well as control the temperature control module to dynamically adjust the temperature.

[0053] Example 3

[0054] This embodiment provides a blood separation method, which is performed using the blood separation device described in Example 2, and includes the following steps:

[0055] (1) Whole blood (heparin anticoagulation, HCT = 45%) was collected from healthy volunteers and divided into 5 groups (n = 5), with 5 mL in each group.

[0056] (2) Start the blood separation device, set the temperature of the temperature control module to 30°C, inject blood into the microchannel from the microchannel inlet of the microfluidic chip at a flow rate of 200 μL / min, set the piezoelectric transducer to output the sound field, the sound wave frequency is 130 kHz, the sound pressure is 50 kPa, and the duration is 25 min. Remove red blood cells and white blood cells, and obtain high-purity plasma matrix from the microchannel outlet;

[0057] At the same time, the optical detection module monitors the concentration and separation effect of red blood cells and platelets in the microchannel of the microfluidic chip in real time; the system control module dynamically analyzes the signals output by the optical detection module and the temperature control module. When an abnormal signal is detected, it controls the piezoelectric transducer to dynamically adjust the sound wave frequency and boost amplitude, or controls the temperature control module to dynamically adjust the temperature.

[0058] Testing revealed a residual red blood cell count of 0.06±0.02% and a residual white blood cell count of <0.01% in the plasma matrix. The platelet activation rate was 4.7±1.2%, the platelet recovery rate was 92.3±3.1%, and the PDGF concentration was 95ng / mL. Whole blood processing was performed simultaneously with blood draw, and the red blood cell separation efficiency fluctuated by less than 2% across different patients, with system temperature rises below 1°C.

[0059] Comparative Example 1

[0060] This comparative example uses traditional centrifugation to separate blood, including the following steps:

[0061] 5 mL of blood was collected using a blood collection tube and centrifuged twice according to the standard preparation protocol for traditional platelet-rich plasma to obtain the upper plasma matrix.

[0062] Testing revealed a residual red blood cell count of 0.62 ± 0.18% in the plasma matrix; a platelet recovery rate of 74.2 ± 5.8%, a platelet activation rate of 4.3 ± 5.1%, a leukocyte recovery rate of 61%, and a leukocyte enrichment of 3.7 times. The total time required for the procedure, including blood draw, was 30 minutes.

[0063] From the comparison between Example 3 and Comparative Example 1, it can be found that when blood is centrifuged using the blood centrifuge provided by the present invention, the platelet recovery rate in the obtained plasma matrix is ​​significantly increased, while the residual white blood cells are significantly reduced, and the obtained plasma matrix has higher purity. In addition, the method of the present invention can process whole blood samples with a hematocrit of 35-50% and is compatible with different blood types and pathological conditions.

[0064] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0065] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments described herein, but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. A microfluidic chip, characterized in that: The microfluidic chip includes a chip substrate, a microchannel arranged inside the chip substrate, and a microchannel inlet and a microchannel outlet. A plurality of grooves are arranged on the bottom wall of the microchannel, and the microchannel is distributed in the shape of an Archimedean spiral.

2. The microfluidic chip according to claim 1, characterized in that The diameter of the groove is 10-100 μm; And / or, the distance between adjacent grooves is 10-500 μm.

3. The microfluidic chip according to claim 1 or 2, characterized in that: The cross-sectional shape of the microchannel is rectangular, the width of the microchannel is 1-10 mm, and the height of the microchannel is 1-10 mm; And / or, the lead of the Archimedean spiral is 1-5 times the width of the microchannel.

4. The microfluidic chip according to any one of claims 1 to 3, characterized in that: The chip substrate is made of polydimethylsiloxane modified glass; And / or, the surface of the microchannel is treated with oxygen plasma.

5. A blood separation device, characterized in that: Comprising the microfluidic chip according to any one of claims 1 to 4, as well as a piezoelectric transducer, an optical detection module, a temperature control module and a system control module; The piezoelectric transducer is used to provide sound waves to the microfluidic chip; The optical detection module is used to monitor the concentration and separation effect of red blood cells and platelets in the microchannel of the microfluidic chip in real time; The temperature control module is used to control the temperature inside the microchannel of the microfluidic chip; The system control module is used to analyze the signals output by the optical detection module and the temperature control module, and control the piezoelectric transducer to dynamically adjust the sound wave frequency and boost amplitude, and control the temperature control module to dynamically adjust the temperature.

6. The blood separation device according to claim 5, characterized in that: The optical detection module includes a dual-wavelength absorbance sensor, and the dual-wavelength absorbance sensor is arranged in the entire microchannel section of the microfluidic chip.

7. A blood separation method, characterized in that: The separation method is performed using the blood separation device according to claim 5 or 6, comprising: Starting the blood separation device, setting the temperature of the temperature control module, injecting blood into the microchannel from the microchannel inlet of the microfluidic chip, setting the piezoelectric transducer to output an acoustic field to remove red blood cells and white blood cells, and obtaining a plasma matrix from the microchannel outlet; At the same time, the optical detection module monitors the concentration and separation effect of red blood cells and platelets in the microchannel of the microfluidic chip in real time. The system control module dynamically analyzes the signals output by the optical detection module and the temperature control module. When an abnormal signal is detected, the piezoelectric transducer is controlled to dynamically adjust the sound wave frequency and boost amplitude, or the temperature control module is controlled to dynamically adjust the temperature.

8. The blood separation method according to claim 7, characterized in that: The flow rate of the blood is 10-500 μL / min.

9. The blood separation method according to claim 7 or 8, characterized in that: The temperature control module is set to a temperature of 4-37°C.

10. The blood separation method according to any one of claims 7 to 9, characterized in that: When a signal abnormality is detected, the piezoelectric transducer is controlled to dynamically adjust the sound wave frequency and the boost amplitude, or the temperature control module is controlled to dynamically adjust the temperature, specifically: When it is detected that the red blood cell migration rate is higher than the set range, the piezoelectric transducer is controlled to increase the sound wave frequency, and the adjustment range of the sound wave frequency is 0-20%; When it is detected that the temperature exceeds the set range, the temperature control module is controlled to increase or decrease the temperature.

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