Microfluidic chip and method for screening early diabetes based on red blood cell morphological changes

Abstract

The present invention discloses a microfluidic chip and method for screening for early-stage diabetes based on changes in red blood cell morphology. The fluid layer of the microfluidic chip is equipped with an hourglass-shaped experimental chamber, with fluid channels connected to both sides of the experimental chamber. The control channels in the control layer integrate multiple sets of control valves that can control the opening and closing of the fluid channels on both sides of the experimental chamber. The combination of control valves alternately switches on and off, generating a controllable reciprocating shear flow field in opposite directions. By controlling the frequency and number of control valves, dynamic shear stress with simultaneous amplitude and frequency regulation can be generated. A small number of red blood cells are introduced into the chip, and the reciprocating shear flow field drives the change in the shape of the red blood cells. By observing the changes in the morphological parameters of red blood cells in the reciprocating flow field, such as volume, deformation index, and hemolysis rate, the present invention establishes a relationship between the changes in red blood cell morphological parameters and the reciprocating flow field, providing a new non-invasive method for early screening of diabetes.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips and relates to a microfluidic chip and a method for screening early diabetes based on red blood cell morphological changes. Background Art

[0002] As a chronic metabolic disease that is difficult to cure, early screening for diabetes is very important. Current early screening mainly relies on blood biochemical markers and physical examinations. The main technologies include fasting plasma glucose (FPG) test and hemoglobin A1c (HbA1c) test. These two methods are widely used, but they both have significant limitations. The test requires a large blood sample, which is invasive and inconvenient for patients. Secondly, these methods have limited accuracy in diagnosing early diabetes. FPG can be affected by short-term factors (food intake, stress), leading to fluctuations in blood sugar. The HbA1c test is more stable over time, but may not be able to detect short-term changes in blood sugar and can be affected by hemoglobin variants or conditions that affect red blood cells. Finding a non-invasive, convenient and fast method for early screening of diabetes has become a key research focus.

[0003] In recent years, researchers have studied non-invasive and non-invasive methods for diabetes screening based on blood flow characteristics and red blood cell deformation index. Nouri et al. used LDI flash imaging technology to detect the reflection of infrared light sources on the skin to evaluate microvascular function and found that the skin vascular reactivity of diabetic patients was significantly reduced. Nieuwenhoff et al. used laser Doppler flowmetry and infrared thermal imaging technology to evaluate the skin blood flow and temperature regulation response of diabetic patients, revealing lower skin blood flow and weaker regulation ability to changes in ambient temperature. However, these methods are affected by changes in ambient temperature and humidity and require special equipment and techniques, making them less suitable for large-scale screening. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the purpose of the present invention is to provide a microfluidic chip and method for screening early-stage diabetes based on changes in red blood cell morphology, thereby achieving mechanical stimulation of red blood cells and addressing the limitations of the prior art in non-invasive, convenient and fast large-scale screening for early-stage diabetes.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A microfluidic chip for screening early-stage diabetes based on changes in red blood cell morphology. The chip consists of a fluid layer, a PDMS film, a control layer, and a glass plate from top to bottom.

[0007] The fluid layer is provided with a sample liquid inlet and a liquid outlet, and a closed microchannel I is formed between the groove flow channel on the bottom surface of the fluid layer and the PDMS film. The closed microchannel I includes a left microfluidic channel, a left microvalve-controlled fluid channel, an experimental chamber, a right microvalve-controlled fluid channel, and a right microfluidic channel that are sequentially connected and located on the same plane. The sample liquid inlet is connected to the left microfluidic channel, and the liquid outlet is connected to the right microfluidic channel. The experimental chamber is a horizontal hourglass-shaped structure;

[0008] The control layer is provided with a control through hole, and a closed microchannel II is formed between the groove flow channel on the top surface of the control layer and the PDMS film. The closed microchannel II can serve as a control valve to control the opening and closing of each channel in the closed microchannel I. The closed microchannel II includes a liquid inlet control channel, a liquid outlet control channel and multiple control channels. The liquid inlet control channel is located directly below the left microfluidic channel and the two are perpendicular to each other. The liquid outlet control channel is located directly below the right microfluidic channel and the two are perpendicular to each other. The multiple control channels are vertically located below the left microvalve control fluid channel and the right microvalve control fluid channel. Each control channel in the closed microchannel II is respectively connected to the control liquid inlet hole.

[0009] The present invention also includes the following technical features:

[0010] Specifically, the experimental chamber is a bilaterally symmetrical horizontal hourglass-shaped structure, wherein the size of the narrow channel at the middle neck is 5 μm to 100 μm, and the size of the experimental chamber is 2500 μm in length×1900 μm in width×50 μm in height.

[0011] Specifically, there are 10 left microvalve-controlled fluid channels that are parallel to each other, with dimensions of 4500 μm in length × 100 μm in width × 25 μm in height; there are 10 right microvalve-controlled fluid channels that are parallel to each other, with dimensions of 4500 μm in length × 100 μm in width × 25 μm in height; the dimensions of the left microfluidic channel and the right microfluidic channel are both 800 μm in length × 200 μm in width × 25 μm in height; the diameters of the sample inlet and outlet holes are both 1000 μm.

[0012] Specifically, after the liquid inlet control channel is filled with water, it can serve as a liquid inlet control valve to control the conduction and blocking of the left micro-flow channel;

[0013] After the liquid outlet control channel is filled with water, it can serve as a liquid outlet control valve to control the conduction and blocking of the right micro-flow channel;

[0014] There are three control channels corresponding to the bottom of the left microvalve control fluid channel, and after the three control channels are filled with water, they can respectively serve as oscillation control valves to control the flow direction of the liquid in the left microvalve control fluid channel;

[0015] There are three corresponding control channels below the right microvalve control fluid channel, and after the three control channels are filled with water, they can respectively serve as oscillation control valves to control the flow direction of the liquid in the right microvalve control fluid channel.

[0016] Specifically, the oscillation control valves are from left to right: first control valve, second control valve, third control valve, fourth control valve, fifth control valve, and sixth control valve;

[0017] The six control valves control the switches of each channel in the fluid layer through the control logic sequence of (1 0 0 1 1 1), (1 1 1 0 0 1), (1 0 0 1 1 1); 1 means the control valve is closed and liquid cannot flow in; 0 means the control valve is open and liquid can flow in, thereby driving the liquid to flow left or right.

[0018] Specifically, the liquid inlet control valve, liquid outlet control valve and oscillation control valve are controlled by controlling the external pressure of the liquid inlet hole; the liquid pressure is driven by the external gas of the liquid inlet hole to cause the control channel to produce spatial deformation, squeeze the PDMS film to block and close the channel in the fluid layer, thereby realizing control of the fluid layer.

[0019] Specifically, the chip amplitude is generated by the flow field changes caused by the switching of control valves on the left and right sides of the experimental chamber; the frequency is regulated by the time interval between the closing and opening of the control valves; and by changing the height of the experimental chamber neck, fluctuating shear force is generated to change the shape of the cells.

[0020] Specifically, multiple experimental chambers and their closed microchannels I can be arranged in parallel in the fluid layer to observe the effects of different shear flows in experimental chambers of different size structures on samples under the regulation of the same dynamic signal; and the number of control valves on the left and right sides of any experimental chamber is the same to form a reciprocating flow field.

[0021] The method for manufacturing the microfluidic chip for screening early diabetes based on red blood cell morphological changes comprises the following steps:

[0022] Step 1: The designed control layer and fluid layer pattern masks are respectively subjected to multiple UV exposure, development, and baking to form two microfluidic silicon wafers; the two microfluidic silicon wafers are respectively a control layer microfluidic silicon wafer and a fluid layer microfluidic silicon wafer;

[0023] Step 2, glue preparation and glue homogenization: weigh the PDMS glue and curing agent, turn on the vacuum degassing mixer, put the weighed PDMS glue and curing agent into the mixer, vacuumize the PDMS glue and curing agent and stir to mix them, control the speed to 2000 rpm for 2 minutes; 1000 rpm for 1 minute to obtain the PDMS mixed glue;

[0024] Step 3, TMCS coating treatment: Place two microfluidic silicon wafers into a volatilization cylinder and add 1 ml of methylchlorosilane;

[0025] Step 4: Slowly pour the PDMS mixed glue obtained in step 2 onto the surface of the control layer microfluidic channel silicon wafer, and spin-coat the PDMS film on the surface of the control layer microfluidic channel silicon wafer at a speed of 500 rpm for 6 seconds and 2200 rpm for 30 seconds. Then, let the control layer microfluidic channel silicon wafer coated with the PDMS film stand for 5 minutes and then place it in an 80°C oven for 15 minutes.

[0026] Step 5: Place the tin foil flat on a plastic Petri dish, place the fluidic layer microfluidic channel silicon wafer in it, gently compact the fluidic layer microfluidic channel silicon wafer, pour in the PDMS mixed glue, and make sure there are no bubbles on the glue on the fluidic layer microfluidic channel silicon wafer. Then, place it in an 80°C oven and let it stand for 2 hours to overnight.

[0027] Step 6, separation: Use a cutting knife to separate the PDMS and the silicon wafer along the outer edge of the fluid layer microfluidic channel silicon wafer and cut it into squares to obtain the PDMS fluid layer;

[0028] Step 7, Plasma treatment: Plasma-treat the PDMS fluid layer and the PDMS film on the control layer. Use a microaligner to place the PDMS fluid layer on the PDMS film on the control layer, plasma bond, and obtain a bonded chip. Bake the aligned chip in an 80°C constant temperature oven for at least 48 hours.

[0029] Step 8, separation: Use a cutting knife to separate the PDMS control layer and the silicon wafer along the outer edge of the control layer microfluidic channel silicon wafer and cut it into squares to obtain the bonded PDMS fluid layer and PDMS control layer;

[0030] Step 9, punching: punch holes in the PDMS fluid layer with a puncher;

[0031] Step 10, Plasma treatment again: Plasma bonding the punched chip to the glass plate, sealing the bottom of the chip, and baking the chip after bonding to the glass plate in a constant temperature oven at 80°C for at least 48 hours to obtain a microfluidic microfluidic chip.

[0032] The method for operating the microfluidic chip for screening early-stage diabetes based on red blood cell morphology changes comprises the following steps:

[0033] Step 1: injecting liquid into the closed microchannel II of the control layer in the chip, and controlling the pressure generated by the deformation in the control channel to control the opening and closing of the closed microchannel I of the fluid layer;

[0034] Step 2: Open the liquid inlet control valve and the liquid outlet control valve;

[0035] Step 3: Use a pipette to insert into the sample inlet hole of the chip and slowly press and inject. When the experimental chamber is filled with cell suspension, close the inlet control valve and the outlet control valve;

[0036] Step 4: Close the first control valve on the left side of the experimental chamber and the sixth control valve on the right side, then simultaneously close the second and third control valves, and simultaneously open the fourth and fifth control valves to allow the liquid to squeeze and flow to the right through the channel and the experimental chamber;

[0037] Step 5: After a period of time, close the fourth control valve and the fifth control valve at the same time, and open the second control valve and the third control valve at the same time, so that the liquid is squeezed and flows to the left through the channel and the experimental chamber;

[0038] Step 6: Repeat steps 4 and 5 to circulate the liquid flow so that the red blood cells are squeezed by the reciprocating shear fluid in the experimental chamber.

[0039] Compared with the prior art, the present invention has the following technical effects:

[0040] 1. Compared with existing microfluidic chips that can perform reciprocating fluid motion, the chip of the present invention can generate a dynamic reciprocating flow field by controlling the number and time of valve groups. It can be used to study the effects of different mechanical oscillations on red blood cells during the reciprocating process, and is suitable for the correlation between cell morphological changes and reciprocating flow fields in early screening for diabetes.

[0041] 2. The chip structure of the present invention is designed with different height restriction structures of structural scale, which can generate fluctuating shear flow, so that the shear force generated at different positions in the chip is different, giving different squeezing forces to the cells.

[0042] 3. The present invention can provide a microenvironment with different dynamic extrusion mechanical signals for the sample in the culture chamber by controlling different frequencies and amplitudes. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the chip structure of the present invention.

[0044] Figure 2 (a) Front view and (b) Left view of the chip of the present invention.

[0045] Figure 3 This is the control logic diagram of the control valve.

[0046] Figure 4 Schematic diagram of the red blood cell sample injection process into the microfluidic chip.

[0047] Figure 5 Schematic diagram of red blood cells passing through the restriction structure in the reciprocating flow field in the microfluidic chip.

[0048] Figure 6 Schematic diagram of a microfluidic chip with six integrated experimental chambers.

[0049] Figure 7Schematic diagram of the changes in the number of spherical cells and ghost cells under different oscillation times.

[0050] Figure 8 Schematic diagram of cell slope statistics of blood samples from healthy volunteers of different ages at 1800 oscillations.

[0051] The meaning of each number in the figure is:

[0052] 1. Sample liquid inlet, 2. Liquid inlet control valve, 3. First control valve, 4. Second control valve, 5. Third control valve, 6. Left microvalve control fluid channel, 7. Fourth control valve, 8. Fifth control valve, 9. Sixth control valve, 10. Experimental chamber, 11. Liquid outlet control valve, 12. Liquid outlet, 13. Left microfluidic channel, 14. PDMS film, 15. Right microfluidic channel; 16. Liquid inlet of liquid inlet and outlet control valve, 17. Liquid inlet of sixth control valve, 18. Liquid inlet of fifth control valve, 19. Liquid inlet of fourth control valve, 20. Liquid inlet of first control valve, 21. Liquid inlet of second control valve, 22. Liquid inlet of third control valve, 23. Glass substrate. DETAILED DESCRIPTION

[0053] The present invention provides a microfluidic chip for screening early-stage diabetes based on changes in red blood cell morphology. By driving liquid through a combination of microvalves within the chip to squeeze red blood cell samples back and forth through a micrometer-scale funnel-shaped channel structure, the fluid pressure and pressure gradient in the channel are determined by the geometric shape. By measuring the morphological changes of red blood cells under shear stress in the flow field, an additional diagnostic parameter is provided for exploring early changes in diabetes.

[0054] Abnormal expression of erythrocyte cytoskeletal proteins in diabetic patients leads to decreased erythrocyte deformability and even abnormal morphology. Agrawal et al. evaluated erythrocyte morphological parameters using digital microscopy, while Chang et al. used a deformability index to assess erythrocyte deformability, demonstrating the important role of abnormal erythrocyte morphological parameters in the development and progression of diabetes. Furthermore, erythrocytes undergo certain shape changes in a flow field. By analyzing these morphological changes under shear stress in a flow field, new indicators of early diabetic changes can be explored. Reverse shear flow is a phenomenon in which fluid motion is induced by the periodic application of positive and reverse shear stresses. This invention constructs a microfluidic chip with integrated microvalves and a symmetrical hyperbolic structure. By controlling the opening and closing of four to eight microvalves, a stable and controllable flow field is generated. A customized MATLAB program periodically changes the state of the valve membrane to generate a reciprocating shear flow field within the chip, continuously changing the shape of erythrocytes. By observing the changes in morphological parameters (such as cell volume, deformability index, and hemolysis rate) of erythrocytes in the reciprocating flow field, a relationship between the changes in erythrocyte morphological parameters and the reciprocating flow field was established. This provides a new non-invasive method for early screening of diabetes, aiming to improve diagnostic accuracy and offering a new perspective for early diagnosis in the field of diabetes. Furthermore, the integration of multiple hyperbolic structures within the chip significantly increases detection throughput, making large-scale diabetes screening possible.

[0055] Specific embodiments of the present invention are given below. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent modifications made on the basis of the technical solution of this application fall within the protection scope of the present invention.

[0056] Example 1:

[0057] like Figures 1 to 4 As shown, this embodiment provides a microfluidic chip for screening early diabetes based on red blood cell morphological changes, which can realize the mechanical stimulation of red blood cells by controllable reciprocating fluid and the mechanical effect of reciprocating shear flow field on red blood cells. Figure 1This is a top view of the chip. The microfluidic chip consists of a fluid layer, a PDMS film 14, a control layer, and a glass substrate 23 from top to bottom. The fluid layer is provided with a sample inlet 1 and a liquid outlet 12. A closed microchannel 1 is formed between the groove flow channel on the bottom surface of the fluid layer and the PDMS film 14. The closed microchannel 1 includes a left microfluidic channel 13, a left microvalve control fluid channel 6, an experimental chamber 10, a right microvalve control fluid channel, and a right microfluidic channel 15, which are sequentially connected and located on the same plane. The sample inlet 1 is connected to the left microfluidic channel 13, and the liquid outlet 12 is connected to the right microfluidic channel 15. The experimental chamber 10 is a horizontal hourglass-shaped structure. A control liquid inlet is provided on the control layer, and a closed microchannel II is formed between the groove flow channel on the top surface of the control layer and the PDMS film 14. The closed microchannel II can be used as a control valve to control the opening and closing of each channel in the closed microchannel I. The closed microchannel II includes a liquid inlet control channel, a liquid outlet control channel and multiple control channels. The liquid inlet control channel is located directly below the left microfluidic channel 13 and the two are perpendicular to each other. The liquid outlet control channel is located directly below the right microfluidic channel 15 and the two are perpendicular to each other. Multiple control channels are located vertically below the left microvalve control fluid channel 6 and the right microvalve control fluid channel. Each control channel in the closed microchannel II is respectively connected to the control liquid inlet.

[0058] The experimental chamber 10 is a bilaterally symmetrical horizontal hourglass-shaped structure used to mechanically stimulate the red blood cell sample at the constricted channel. The narrow channel at the middle constriction ranges from 5 μm to 100 μm. The constricted structure of the experimental chamber 10 is regulated at different scales to generate fluctuating squeezing pressure. The dimensions of the experimental chamber 10 are 2500 μm long, 1900 μm wide, and 50 μm high.

[0059] There are 10 left microvalve control fluid channels 6 and they are connected in parallel with each other, and their dimensions are 4500 μm long × 100 μm wide × 25 μm high; there are 10 right microvalve control fluid channels and they are connected in parallel with each other, and their dimensions are 4500 μm long × 100 μm wide × 25 μm high;

[0060] The dimensions of the left microfluidic channel 13 and the right microfluidic channel 15 are both 800 μm long × 200 μm wide × 25 μm high;

[0061] The diameters of the sample liquid inlet 1 and the liquid outlet 12 are both 1000 μm.

[0062] After the liquid inlet control channel is filled with water, it can serve as a liquid inlet control valve 2 to control the conduction and blocking of the left micro-flow channel 13;

[0063] After the liquid outlet control channel is filled with water, it can serve as a liquid outlet control valve 11 to control the conduction and blocking of the right micro-channel 15;

[0064] There are three corresponding control channels below the left microvalve control fluid channel 6. After the three control channels are filled with water, they can serve as oscillation control valves to control the flow direction of the liquid in the left microvalve control fluid channel 6.

[0065] There are three corresponding control channels below the right microvalve control fluid channel, and after the three control channels are filled with water, they can respectively serve as oscillation control valves to control the flow direction of the liquid in the right microvalve control fluid channel;

[0066] Furthermore, the oscillation control valves are from left to right: the first control valve 3, the second control valve 4, the third control valve 5, the fourth control valve 7, the fifth control valve 8, and the sixth control valve 9. The six control valves control the switches of each channel of the fluid layer through the control logic sequence of (1 0 0 1 1 1), (11 1 0 01), and (1 0 0 1 1 1), where 1 means the control valve is closed and the liquid cannot flow in, and 0 means the control valve is open and the liquid can flow in, driving the liquid to flow left or right.

[0067] The aforementioned inlet control valve 2, outlet control valve 11, and oscillation control valve are controlled by controlling the pressure (10-30 psi) applied to the inlet. This pressure, driven by the gas applied to the inlet, causes the control channel to deform spatially, squeezing the PDMS film 14 to block and close the channel within the fluid layer, thereby achieving control of the fluid layer.

[0068] Each control channel in the closed microchannel II is connected to the control liquid inlet hole respectively. Specifically, the liquid inlet control channel and the liquid outlet control channel are both connected to the liquid inlet hole 16 of the inlet and outlet control valve. The control channels corresponding to the first control valve 3, the second control valve 4, the third control valve 5, the fourth control valve 7, the fifth control valve 8 and the sixth control valve 9 are respectively connected to the first control valve liquid inlet hole 20, the second control valve liquid inlet hole 21, the third control valve liquid inlet hole 22, the fourth control valve liquid inlet hole 19, the fifth control valve liquid inlet hole 18 and the sixth control valve liquid inlet hole 17.

[0069] The opening and closing parts of the chip of this invention are control layer control valves. The gas drives the liquid pressure (17psi to 45psi) to cause spatial deformation of the control valve channel, thereby realizing the control of the fluid layer microchannel switch.

[0070] The input of dynamic mechanical signals includes the regulation of amplitude, frequency and different confinement sizes of the experimental cavity 10.

[0071] The amplitude is generated by the flow field changes caused by the switching of the control valves on the left and right sides of the experimental chamber 10. The control valves in column 1 correspond to 5nL liquid squeezing, the control valves in column 2 realize 10nL liquid squeezing, and the control valves in column 3 realize 15nL liquid squeezing.

[0072] The frequency is controlled by the time interval between closing and opening of the control valve.

[0073] The middle of the experimental chamber 10 is an arc-shaped structure of different scales. The height of the neck of the experimental chamber 10 is changed to generate fluctuating shear force to change the shape of the cells.

[0074] like Figure 6 The fluid layer consists of 1-10 parallel experimental chambers 10, used to observe the effects of different shear flows on samples under the control of the same dynamic signal in experimental chambers 10 with different defined dimensions. An equal number of micro-control valves are designed on the left and right sides of each experimental chamber 10 to create a reciprocating flow field.

[0075] Example 2:

[0076] This embodiment provides a method for manufacturing a microfluidic chip for screening early diabetes based on red blood cell morphology changes, comprising the following steps:

[0077] Step 1: The designed control layer and fluid layer pattern masks are respectively made into two microfluidic silicon wafers through multiple UV exposure, development, and baking; the two microfluidic silicon wafers are the control layer microfluidic silicon wafer and the fluid layer microfluidic silicon wafer; specifically, during the photolithography process, the geometric dimensions and configuration of the microchannels are derived from the original mask pattern, so the primary task of photolithography is to draw the two-dimensional pattern of the microchannels on the mask; the pattern drawing can be completed by using AutoCAD computer drawing software; the microfluidic chip template is made using contact photolithography technology; and then the microchannel is completed through multiple UV exposure, development, and baking. Then the liquid PDMS material is poured onto the silicon wafer by pouring, and after curing, an elastic PDMS microchannel groove is obtained, specifically:

[0078] Step 2, prepare and evenly distribute the glue: Turn on the balance to weigh the glue, the mass ratio of glue A (PDMS): glue B (curing agent, such as silane coupling agent) is 10:1; the larger the ratio of glue A to glue B, the softer the prepared glue; evenly distribute the glue: Turn on the vacuum degassing mixer, put in the weighed glue, vacuum and stir to mix the glue, 2000 rpm, 2 minutes; 1000 rpm, 1 minute, to obtain PDMS mixed glue.

[0079] Step 3, TMCS coating treatment: Place two microfluidic silicon wafers in a volatilization cylinder and add 1 ml of methylchlorosilane (TMCS) for about 15 minutes.

[0080] Step 4, prepare the control layer and PDMS film: slowly pour the PDMS mixed glue with a total weight of 10g obtained in step 2 onto the surface of the control layer microfluidic channel silicon wafer, spin-coat the PDMS film on the surface of the control layer microfluidic channel silicon wafer at a speed of 500rpm for 6s; 2200rpm for 30s, then let the control layer microfluidic channel silicon wafer coated with the PDMS film stand for 5mins and then put it into an 80℃ oven for 15mins.

[0081] Step 5, prepare the fluid layer: pouring glue: spread the tin foil flat in a 4-inch plastic culture dish, put the fluid layer microfluidic channel silicon wafer in, gently compact the fluid layer microfluidic channel silicon wafer and pour 50g of PDMS mixed glue, making sure there are no bubbles on the glue on the fluid layer microfluidic channel silicon wafer; degassing: place the culture dish after pouring glue in a vacuum box to remove bubbles (negative pressure 0.8), and then put it in an 80℃ oven and let it stand for 2 hours to overnight.

[0082] Step 6, separation: Use a cutting knife to carefully separate the PDMS and the silicon wafer along the outer edge of the fluid layer microfluidic channel silicon wafer and cut it into squares. Cut neatly to obtain the PDMS fluid layer.

[0083] Step 7, Plasma treatment: Plasma treatment of the PDMS fluid layer and the PDMS film on the control layer. Alignment: Use a microaligner to place the PDMS fluid layer on the PDMS film on the control layer, plasma bonding to obtain a bonded chip. Baking: Bake the aligned chip in a constant temperature oven at 80°C for at least 48 hours.

[0084] Step 8, separation: Use a cutting knife to carefully separate the PDMS control layer and the silicon wafer along the outer edge of the control layer microfluidic channel silicon wafer and cut them into squares. Cut them neatly to obtain the bonded PDMS fluid layer and PDMS control layer.

[0085] Step 9, punching: Use a puncher to punch holes in the PDMS fluid layer, paying attention to the location and hole size (0.7 mm).

[0086] Step 10: Plasma treatment again: plasma bond the punched chip to the glass plate, seal the bottom of the chip, and bake: bake the chip after bonding to the glass plate in a constant temperature oven at 80°C for at least 48 hours to obtain a microfluidic microfluidic chip.

[0087] Example 3:

[0088] This embodiment provides an operating method of a microfluidic chip for screening early-stage diabetes based on red blood cell morphology changes, comprising the following steps:

[0089] Step 1: injecting liquid into the closed microchannel II of the control layer in the chip, and controlling the pressure generated by the deformation in the control channel to control the opening and closing of the closed microchannel I of the fluid layer;

[0090] Step 2: Open the liquid inlet control valve and the liquid outlet control valve;

[0091] Step 3: Use a pipette to insert into the sample inlet hole of the chip and slowly press and inject. When the experimental chamber is filled with cell suspension, close the inlet control valve and the outlet control valve;

[0092] Step 4: Close the first control valve on the left side of the experimental chamber and the sixth control valve on the right side, then close the second control valve and the third control valve at the same time, and open the fourth control valve and the fifth control valve at the same time, so that the liquid is squeezed and flows to the right through the channel and the experimental chamber.

[0093] Step 5: After a period of time, close the fourth control valve and the fifth control valve at the same time, and open the second control valve and the third control valve at the same time, so that the liquid is squeezed and flows to the left through the channel and the experimental chamber.

[0094] Step 6: Repeat steps 4 and 5 to circulate the liquid flow so that the red blood cells are squeezed by the reciprocating shear fluid in the experimental chamber.

[0095] Specifically, the six control valves of the oscillation control valve control the opening and closing of each channel in the fluid layer through the control logic sequence of (1 0 0 1 1 1), (1 1 1 0 0 1), and (1 0 0 11 1), where 1 indicates that the control valve is closed and liquid cannot flow in, and 0 indicates that the control valve is open and liquid can flow in, driving the liquid to the left or right. The first and sixth control valves ensure that the total amount of reciprocating liquid remains unchanged. The second and third control valves are closed, and the liquid is squeezed to the right at the same time. The entire 10nL cell suspension is forced to pass through the narrow channel of the hyperbolic cavity to the right. Then the second and third control valves are opened, and the fourth and fifth control valves are closed at the same time. The liquid is squeezed to the left at the same time. The entire 10nL cell suspension is forced to pass through the narrow channel of the hyperbolic cavity to the left. This process is repeated for reciprocating drive.

[0096] For the four valves, the peak flow rate at the center of the hyperbolic experimental cavity is about 15.23 mm / s, and the two sides of the experimental cavity are squeezed alternately, such as Figure 5 The interval between valve operation steps is 50 milliseconds, and the time required for one cycle is 200 milliseconds. Six hyperbolic experimental chambers of different sizes are integrated into the same microfluidic chip to improve the throughput of blood testing, such as Figure 6 .

[0097] In this microfluidic chip, samples from healthy volunteers were used for testing. After 500 reciprocating oscillations, deformation of red blood cells, spherocytosis, was observed. The morphological changes were caused by the destruction of the cytoskeleton. When the number of oscillations was increased to 2000 times, the repeated mechanical stress caused the rupture of the red blood cell membrane, causing hemolysis. These cells are basically empty shells of red blood cell membranes, lacking their internal contents, and are called ghost cells, such as Figure 7 The number of ghost cells increased linearly with mechanical stimulation, and the slope showed correlation with physiological and pathological conditions.

[0098] In a statistical test of samples from 20 healthy volunteers, it was observed that there was no significant difference in glucose disposal for most samples over 24 hours, e.g. Figure 8 , but one 40-year-old woman and three men over 38 years old showed a lag time that was approximately 300-fold shorter. These results suggest that the response of red blood cells to pathological conditions (i.e., high glucose levels) may vary from person to person, but age is an important factor, with the slope being significantly higher in the elderly than in the young. These results indicate that age significantly affects the mechanical elasticity of red blood cells, with the elderly showing a faster onset of hemolysis under mechanical stress.

[0099] These experiments demonstrated a clear correlation between red blood cell deformation and age. Studies have also shown differences in stiffness between diabetic and healthy subjects, highlighting the potential of using red blood cell mechanical properties as a diagnostic tool for early diabetes detection. The ability of microfluidic chips to generate controlled shear flow fields and measure red blood cell morphological changes offers a promising high-throughput diagnostic solution for large-scale clinical screening.

Claims

1. A microfluidic chip for screening early-stage diabetes based on red blood cell morphological changes, characterized in that: The microfluidic chip comprises a fluid layer, a PDMS film (14), a control layer and a glass substrate (23) from top to bottom; The fluid layer is provided with a sample liquid inlet (1) and a liquid outlet (12), and a closed microchannel I is formed between the groove flow channel on the bottom surface of the fluid layer and the PDMS film (14). The closed microchannel I includes a left microfluidic channel (13), a left microvalve control fluid channel (6), an experimental chamber (10), a right microvalve control fluid channel and a right microfluidic channel (15) which are connected in sequence and located on the same plane. The sample liquid inlet (1) is connected to the left microfluidic channel (13), and the liquid outlet (12) is connected to the right microfluidic channel (15). The experimental chamber (10) is a horizontal hourglass-shaped structure. The control layer is provided with a control through hole, and a closed microchannel II is formed between the groove flow channel on the top surface of the control layer and the PDMS film (14). The closed microchannel II can be used as a control valve to control the opening and closing of each channel in the closed microchannel I. The closed microchannel II includes a liquid inlet control channel, a liquid outlet control channel and a plurality of control channels. The liquid inlet control channel is located directly below the left microfluidic channel (13) and the two are perpendicular to each other. The liquid outlet control channel is located directly below the right microfluidic channel (15) and the two are perpendicular to each other. The plurality of control channels are vertically located below the left microvalve control fluid channel (6) and the right microvalve control fluid channel. Each control channel in the closed microchannel II is connected to the control liquid inlet hole respectively. After the liquid inlet control channel is filled with water, it can serve as a liquid inlet control valve (2) to control the conduction and blocking of the left micro-flow channel (13); After the liquid outlet control channel is filled with water, it can serve as a liquid outlet control valve (11) to control the conduction and blocking of the right micro-flow channel (15); There are three corresponding control channels below the left microvalve control fluid channel (6), and after the three control channels are filled with water, they can respectively serve as oscillation control valves to control the flow direction of the liquid in the left microvalve control fluid channel (6); There are three corresponding control channels below the right microvalve control fluid channel, and after the three control channels are filled with water, they can respectively serve as oscillation control valves to control the flow direction of the liquid in the right microvalve control fluid channel; The oscillation control valves are, from left to right, the first control valve (3), the second control valve (4), the third control valve (5), the fourth control valve (7), the fifth control valve (8), and the sixth control valve (9); The six control valves control the switches of each channel in the fluid layer through the control logic sequence of (1 0 0 1 1 1), (1 1 1 0 0 1), (1 0 0 1 1 1); 1 means the control valve is closed and liquid cannot flow in; 0 means the control valve is open and liquid can flow in, thereby driving the liquid to flow left or right.

2. The microfluidic chip for screening early diabetes based on red blood cell morphology changes according to claim 1, characterized in that: The experimental chamber (10) is a horizontal hourglass-shaped structure that is bilaterally symmetrical, wherein the size of the narrow channel at the middle necking portion is 5 μm to 100 μm, and the size of the experimental chamber (10) is 2500 μm in length × 1900 μm in width × 50 μm in height.

3. The microfluidic chip for screening early diabetes based on red blood cell morphology changes according to claim 1, characterized in that: There are 10 left microvalve control fluid channels (6) which are parallel to each other and have dimensions of 4500 μm in length × 100 μm in width × 25 μm in height; There are 10 right microvalve-controlled fluid channels that are parallel to each other, with dimensions of 4500 μm in length × 100 μm in width × 25 μm in height; the dimensions of the left microfluidic channel (13) and the right microfluidic channel (15) are both 800 μm in length × 200 μm in width × 25 μm in height; the diameters of the sample inlet (1) and outlet (12) are both 1000 μm.

4. The microfluidic chip for screening early diabetes based on red blood cell morphology changes according to claim 1, characterized in that: The liquid inlet control valve (2), the liquid outlet control valve (11) and the oscillation control valve are controlled by controlling the pressure external to the liquid inlet hole; the liquid pressure is driven by the gas external to the liquid inlet hole to cause the control channel to generate spatial deformation, squeeze the PDMS film (14), block and close the channel in the fluid layer, and thus achieve control of the fluid layer.

5. The microfluidic chip for screening early diabetes based on red blood cell morphology changes according to claim 1, characterized in that: The chip amplitude is generated by the flow field change caused by the switching of the control valves on the left and right sides of the experimental chamber (10); the frequency is regulated by the time interval between the closing and opening of the control valves; and by changing the height of the neck of the experimental chamber (10), a fluctuating shear force is generated to change the shape of the cells.

6. The microfluidic chip for screening early diabetes based on red blood cell morphology changes according to claim 1, characterized in that: A plurality of experimental chambers (10) and their closed microchannels I are arranged in parallel in the fluid layer to observe the effects of different shear flows in experimental chambers (10) of different sizes and structures on the sample under the control of the same dynamic signal; and the number of control valves on the left and right sides of any experimental chamber (10) is the same to form a reciprocating flow field.

7. The method for preparing a microfluidic chip for screening early diabetes based on red blood cell morphology changes according to claim 1, characterized in that: The following steps are involved: Step 1: The designed control layer and fluid layer pattern masks are respectively subjected to multiple UV exposure, development, and baking to form two microfluidic silicon wafers; the two microfluidic silicon wafers are respectively a control layer microfluidic silicon wafer and a fluid layer microfluidic silicon wafer; Step 2, glue preparation and glue homogenization: weigh the PDMS glue and curing agent, turn on the vacuum degassing mixer, put the weighed PDMS glue and curing agent into the mixer, vacuumize the PDMS glue and curing agent and stir to mix them, control the speed to 2000 rpm for 2 minutes and 1000 rpm for 1 minute to obtain the PDMS mixed glue; Step 3, TMCS coating treatment: Place two microfluidic silicon wafers into a volatilization cylinder and add 1 ml of methylchlorosilane; Step 4: Slowly pour the PDMS mixed glue obtained in step 2 onto the surface of the control layer microfluidic channel silicon wafer, and spin-coat the PDMS film on the surface of the control layer microfluidic channel silicon wafer at a speed of 500 rpm for 6 seconds and 2200 rpm for 30 seconds. Then, let the control layer microfluidic channel silicon wafer coated with the PDMS film stand for 5 minutes and then place it in an 80°C oven for 15 minutes. Step 5: Place the tin foil flat on a plastic Petri dish, place the fluidic layer microfluidic channel silicon wafer in it, gently compact the fluidic layer microfluidic channel silicon wafer, pour in the PDMS mixed glue, and make sure there are no bubbles on the glue on the fluidic layer microfluidic channel silicon wafer. Then, place it in an 80°C oven and let it stand for 2 hours to overnight. Step 6, separation: Use a cutting knife to separate the PDMS and the silicon wafer along the outer edge of the fluid layer microfluidic channel silicon wafer and cut it into squares to obtain the PDMS fluid layer; Step 7, Plasma treatment: Plasma-treat the PDMS fluid layer and the PDMS film on the control layer. Use a microaligner to place the PDMS fluid layer on the PDMS film on the control layer, plasma bond, and obtain a bonded chip. Bake the aligned chip in an 80°C constant temperature oven for at least 48 hours. Step 8, separation: Use a cutting knife to separate the PDMS control layer and the silicon wafer along the outer edge of the control layer microfluidic channel silicon wafer and cut it into squares to obtain the bonded PDMS fluid layer and PDMS control layer; Step 9, punching: punch holes in the PDMS fluid layer with a puncher; Step 10, Plasma treatment again: Plasma bonding the punched chip to the glass plate, sealing the bottom of the chip, and baking the chip after bonding to the glass plate in a constant temperature oven at 80°C for at least 48 hours to obtain a microfluidic microfluidic chip.

8. The method for operating the microfluidic chip for screening early diabetes based on red blood cell morphology changes according to claim 1, characterized in that: The following steps are involved: Step 1: injecting liquid into the closed microchannel II of the control layer in the chip, and controlling the pressure generated by the deformation in the control channel to control the opening and closing of the closed microchannel I of the fluid layer; Step 2: Open the liquid inlet control valve and the liquid outlet control valve; Step 3: Use a pipette to insert into the sample inlet hole of the chip and slowly press and inject. When the experimental chamber is filled with cell suspension, close the inlet control valve and the outlet control valve; Step 4: Close the first control valve on the left side of the experimental chamber and the sixth control valve on the right side, then simultaneously close the second and third control valves, and simultaneously open the fourth and fifth control valves to allow the liquid to squeeze and flow to the right through the channel and the experimental chamber; Step 5: After a period of time, close the fourth control valve and the fifth control valve at the same time, and open the second control valve and the third control valve at the same time, so that the liquid is squeezed and flows to the left through the channel and the experimental chamber; Step 6: Repeat steps 4 and 5 to circulate the liquid flow so that the red blood cells are squeezed by the reciprocating shear fluid in the experimental chamber.

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