Red blood cell mechanical fatigue damage and mechanical characterization integrated chip

Abstract

The invention discloses an integrated chip for mechanical fatigue damage and mechanical characterization of red blood cells, and particularly relates to the crossing field of a micro-fluidic technology and cell mechanics. Comprising a substrate, and a PDMS functional layer is arranged on the substrate; the substrate and the PDMS functional layer are tightly bonded to form a composite micro-channel system with bionic flow field characteristics, the composite micro-channel system is composed of a fatigue loading structure, a fluid buffer structure and a bionic detection structure which are sequentially communicated, the fatigue loading structure is composed of straight micro-channels and arc-shaped flow channels which are alternately communicated, the fluid buffer structure adopts a taper gradually-shrinking design, and the bionic detection structure adopts a bionic detection structure. A plurality of orthogonally arranged micro-column arrays are arranged in the bionic detection structure and the fatigue loading structure. By adopting the technical scheme, the problems of mechanical environment distortion and insufficient observation resolution of a traditional method are solved, and a standardized platform is provided for mechanical property evaluation and clinical pathological diagnosis of red blood cells under health and fatigue damage.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology and cell mechanics, and particularly relates to a red blood cell mechanical fatigue damage and mechanical characterization integrated chip. BACKGROUND

[0002] Microfluidic technology has become an important tool for cell mechanics research due to its miniaturization, high sensitivity, and rapid analysis, and has shown unique advantages in the field of red blood cell mechanical property characterization. Red blood cells, as the core functional carriers of the human circulatory system, their mechanical properties (such as deformability, membrane elasticity) directly determine the efficiency of microcirculation and oxygen transport capacity. Studies have shown that in pathological conditions such as diabetic vasculopathy and sickle cell anemia, red blood cells are damaged due to long-term exposure to abnormal shear stress (such as high-frequency compression of capillaries), leading to membrane skeleton protein damage, morphological distortion, and oxygen carrying function decline. This "mechanical fatigue" phenomenon is a key indicator of disease progression. However, existing red blood cell fatigue research techniques have the following bottlenecks:

[0003] Mechanical loading distortion: Traditional centrifugal oscillation method (shear range 0.1-10 Pa) cannot reproduce the physiological mechanical environment of capillary pulsation (1.2 Hz, pulsatile pressure 8.3 kPa) due to single loading direction and large time domain fluctuations (CV>15%);

[0004] Dynamic observation disconnection: Static culture combined with end-point electron microscopy detection can only obtain the morphology after fatigue (resolution>200 nm), and cannot track the compression-relaxation dynamic process in real time (time resolution>1 s);

[0005] Multi-parameter analysis fragmentation: Rheometer, atomic force microscope and other devices require step-by-step operation, resulting in time and space mismatch between mechanical parameters (DI, τ) and flow field data (velocity, pressure) (synchronization error>500 ms).

[0006] Although microfluidic technology has achieved precise shear force control (error ±5%) through microchannel topology optimization (such as serpentine, contraction-expansion structure) in recent years, it is still limited to single-mode functions (such as mechanical loading or static detection), and lacks integrated design for "fatigue accumulation-flow field adaptation-in situ detection" whole chain. Therefore, developing a "mechanical loading-flow field adaptation-in situ detection" three-mode integrated architecture microfluidic chip system that can couple dynamic mechanical simulation, cross-flow field flow rate adaptation, and multi-parameter synchronous capture is the key to breaking through the bottleneck of red blood cell fatigue mechanism research and clinical diagnosis technology. SUMMARY

[0007] The present application aims to provide a red blood cell mechanical fatigue damage and mechanical characterization integrated chip, which solves the problems of mechanical environment distortion and insufficient observation resolution in traditional methods.

[0008] In order to achieve the above object, the technical scheme of the present application is as follows: a red blood cell mechanical fatigue damage and mechanical characterization integrated chip, comprising a substrate, wherein the substrate is provided with a PDMS functional layer; the substrate and the PDMS functional layer are tightly bonded to form a composite microchannel system with a biomimetic flow field characteristic, the composite microchannel system is composed of a fatigue loading structure, a fluid buffer structure and a biomimetic detection structure which are sequentially communicated, the fatigue loading structure is composed of straight microchannels and arc-shaped flow channels which are alternately communicated, the fluid buffer structure is designed with a tapering structure, one end of the fluid buffer structure with a smaller size is communicated with the fatigue loading structure, and the other end of the fluid buffer structure with a larger size is communicated with the biomimetic detection structure, and a plurality of microcolumn arrays arranged in quadrature are arranged in the fatigue loading structure and the biomimetic detection structure.

[0009] Further, the size of the microcolumn array in the fatigue loading structure is 4 μm x 5 μm x 15 μm, and the size of the microcolumn array in the biomimetic detection structure is 5 μm x 5 μm x 30 μm.

[0010] Further, the flow channel design of the fatigue loading structure follows the modified Womersley pulsatile flow model, and the pressure gradient equation is expressed as:

[0011]

[0012] In the formula, μ = 1.2 x 10 -3 Pa·s, is the viscosity of PBS solution at 37 DEG C, Q = 0.4 μL / min, is the volume flow rate, corresponding to the inlet flow rate of 15 mm / s, ρ = 1030 kg / m 3 , is the density of red blood cell suspension, and the gradient flow resistance network is constructed by the alternately arranged straight microchannel array and arc-shaped flow channel.

[0013] Through the above setting, the straight microchannel segment generates a basic pressure gradient (6.2 kPa / mm); the arc-shaped flow channel enhances the local resistance through the secondary flow effect; and the flow channel is alternately arranged to form a resonance cavity effect, so that the natural frequency is locked at 1.2 Hz ± 0.1 Hz.

[0014] Further, the inlet flow rate design method of the fatigue loading structure is as follows:

[0015] S1, inlet section flow resistance optimization: a progressive contraction inlet is adopted, and the contraction angle 5 DEG is determined by numerical simulation of Navier-Stokes equation, so that the inlet pressure loss is reduced to 0.8 kPa;

[0016] S2, flow control parameters of the straight microchannel array: single straight channel flow resistance:

[0017]

[0018] The number of parallel channels N=32 is adopted, and the flow field uniformity simulation is used to determine;

[0019] S3, curvature radius gradient design: arranging the curvature radius of the arc-shaped flow channel according to an exponential law, and constructing a flow resistance differential:

[0020]

[0021] Through the ANSYS Fluent multi-physical field coupling simulation verification, the flow velocity fluctuation coefficient can be controlled within ±2.1%, and the outlet pulsatile pressure amplitude measured by the micro pressure sensor is 8.3kPa±0.5kPa, which proves that it can accurately reproduce the mechanical environment of capillary pulsation.

[0022] Further, the cross-sectional area of the flow channel of the fluid buffer structure follows an exponential decay law along the flow direction:

[0023] A(x)=A0·e -βx (0≤x≤L);

[0024] In the formula, A0 is the initial cross-sectional area of the inlet, β is the cross-sectional area decay coefficient optimized by CFD simulation, and L is the total length of the transition unit. By coupling with the Navier-Stokes equation, the flow velocity field distribution equation is derived:

[0025]

[0026] Where v0 is the inlet flow velocity, and α is the flow velocity decay gradient coefficient, which is obtained by joint calibration of the rheological properties of red blood cell suspension and the wall slip boundary condition.

[0027] Through the above setting, the tapering fluid buffer structure can realize the linear decay of flow velocity from 15mm / s to 0.4mm / s, matching the physiological flow velocity of 0.3-0.7mm / s in human capillaries.

[0028] Further, the use method of the integrated chip is as follows: injecting red blood cells into the chip, guiding the directional flow of red blood cells in the chip by using a suction pump, and synchronously acquiring the flow velocity, deformation index and shape relaxation time of red blood cells by a high-speed microscopic imaging system and an optical signal acquisition structure arranged outside the chip.

[0029] Further, the surface of the bionic detection structure is provided with a periodically distributed nano-grating structure, the grating period is 200nm±20nm, and the depth is 50nm±5nm, which satisfies the light scattering enhancement condition:

[0030]

[0031] Where λ=500-600nm is the imaging wavelength, η PDMS= 1.43 is the refractive index of the material, and A is the grating period.

[0032] Through the above design, the micro-column array detection area cooperates with the high-speed microscopic imaging system to realize sub-pixel resolution of red blood cell morphological parameters.

[0033] Further, the optical signal acquisition structure includes a dark field illumination module, a polarization analysis module and a synchronous control unit, the dark field illumination module and the polarization analysis module adopt a coaxial spatial layout design; the specific method is as follows: the dark field illumination module is integrated in the periphery of the objective lens of the high-speed microscopic imaging system in a coaxial ring structure, the ring LED array forms a 60°±5° oblique illumination light path with the objective lens; the polarization analysis module includes a rotatable analyzer and a four-quadrant photodetector, and is embedded in the rear end of the imaging light path, located at the sensor focal plane conjugate position of the high-speed camera; the synchronous control unit synchronously triggers illumination and imaging at a frame frequency of 1000fps, based on the optical flow equation:

[0034] I x u+I y v+I t = 0.

[0035] The red blood cell velocity field distribution is calculated and the deformation index and the morphological relaxation time are synchronously output.

[0036] Compared with the prior art, the beneficial effects of the present scheme are:

[0037] 1. The present scheme adopts a double-mode flow field coupling system of S-shaped fatigue loading structure and fluid buffer structure, breaks through the mechanical loading bottleneck of traditional single-channel chips, and realizes quantitative characterization of the viscoelastic mechanical properties caused by red blood cell fatigue damage.

[0038] 2. The present scheme adopts a 5100 times / cycle directional extrusion design, so that the mechanical fatigue equivalent simulation of the red blood cell stage life cycle can be completed in a single detection, the cumulative effect of red blood cell fatigue is realized, and the detection efficiency is improved by more than 90%.

[0039] 3. The present scheme adopts a taper tapering fluid buffer structure, realizes linear attenuation of flow rate from 15mm / s to 0.4mm / s, matches the physiological flow rate (0.3-0.7mm / s) in human capillaries, and solves the defocusing problem of cross-domain cell tracking.

[0040] 4. The light scattering enhancement effect of the nano-grating structure in the present scheme improves the optical detection sensitivity by 15-20 times, and achieves synchronous capture of sub-cell level mechanical parameters; combined with the high-speed microscopic imaging system, the red blood cell flow velocity, deformation index and morphological relaxation time and other multi-dimensional mechanical parameters can be synchronously captured.

[0041] 5、The scheme realizes the detection flux of 104 cells / min through the chip integration architecture, which is significantly better than the traditional flow cytometer (flux of 103 cells / min), can batch evaluate the red blood cell mechanical phenotype, and provides the red blood cell mechanical quantitative standard (in line with the ISO 13485 clinical equipment specification) for the clinical red blood cell mechanical performance evaluation and the early diagnosis of diseases such as diabetes and sickle cell anemia. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a use method schematic diagram of a red blood cell mechanical fatigue damage and mechanical characterization integrated chip of the present application;

[0043] Figure 2 is a top view of a red blood cell mechanical fatigue damage and mechanical characterization integrated chip of the present application;

[0044] Figure 3 is a top view structural schematic diagram of a composite microchannel system in the embodiment;

[0045] Figure 4 is Figure 3 is a local enlarged view of A in FIG. 1;

[0046] Figure 5 is a scanning electron microscope characterization morphology diagram for different fatigue morphologies of red blood cells;

[0047] Figure 6 is Figure 5 is a direct flow deformation diagram of red blood cells at different fatigue stages in the microchannel of the biomimetic detection structure;

[0048] Figure 7 is a fatigue morphology distribution histogram for quantitatively characterizing the mechanical fatigue damage proportion of a red blood cell population proposed by the present application;

[0049] Figure 8 is a scatter plot distribution diagram of flow velocity for quantifying the flow deformation characteristics of red blood cells of different fatigue morphologies proposed by the present application;

[0050] Figure 9 is a scatter plot distribution diagram of deformation index for quantifying the deformation performance of red blood cells of different fatigue morphologies proposed by the present application;

[0051] Figure 10 is a flow chart of a manufacturing method of a red blood cell mechanical fatigue damage and mechanical characterization integrated chip based on multi-stage flow field regulation of the present application.

[0052] The reference signs in the drawings of the specification include: substrate 1, PDMS functional layer 2, composite microchannel system 3, fatigue loading structure 4, fluid buffer structure 5, biomimetic detection structure 6, straight microchannel 7, arc-shaped flow channel 8. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below through specific embodiments:

[0054] Example

[0055] like Figures 1 to 10 As shown, an integrated chip for mechanical fatigue damage and mechanical characterization of red blood cells includes a substrate 1 on which a PDMS functional layer 2 is provided; the substrate 1 and the PDMS functional layer 2 are tightly bonded to form a composite microchannel system 3 with bionic flow field characteristics, and the composite microchannel system 3 consists of a fatigue loading structure 4, a fluid buffer structure 5 and a bionic detection structure 6 that are connected in sequence.

[0056] The fatigue loading structure 4 is composed of alternating straight microchannels 7 and curved flow channels 8. In this embodiment, the size of the microcolumn array in the fatigue loading structure 4 is 4μm×5μm×15μm. The microcolumn array is distributed in the straight microchannel 7 and the curved flow channel 8 to ensure that the red blood cells maintain disc-shaped directional motion during high-speed flow. The curvature radius gradients on the inner and outer sides of the curved flow channel 8 are 50 and 210μm, respectively. The gradient design achieves an equivalent extrusion frequency of 5100 times / cycle for red blood cells, simulating the fatigue accumulation effect of red blood cells in vivo when flowing with blood. At the same time, the flow channel design of the fatigue loading structure 4 follows the modified Womersley pulsating flow model, and its pressure gradient equation is expressed as:

[0057]

[0058] Where: μ = 1.2 × 10 -3 Pa·s is the viscosity of PBS solution at 37°C, Q = 0.4 μL / min is the volume flow rate, corresponding to an inlet flow rate of 15 mm / s, and ρ = 1030 kg / m 3 , is the density of the red blood cell suspension, and a gradient flow resistance network is constructed by alternating an array of straight microchannels 7 and curved flow channels 8. The 7 sections of straight microchannels generate a basic pressure gradient (6.2kPa / mm), and the curved flow channel 8 enhances the local resistance through the secondary flow effect; the alternating arrangement of the flow channels forms a resonant cavity effect, locking the natural frequency at 1.2Hz±0.1Hz. Through the flow resistance gradient design, when the inlet flow rate is 15mm / s, the outlet pulsating pressure amplitude reaches 8.3kPa±0.5kPa, and the frequency matches the human capillary pulsation frequency of 1.2Hz±0.1Hz. Among them, the design method of the inlet flow velocity of the fatigue loading structure 4 is as follows:

[0059] S1. Optimization of inlet flow resistance: A progressively contracting inlet was used, and the optimal contraction angle of 5° was determined through numerical simulation of the Navier-Stokes equation, reducing the inlet pressure loss to 0.8 kPa (a 67% reduction compared to a right-angle inlet).

[0060] S2, flow control parameters of the straight microchannel 7 array: single straight channel flow resistance:

[0061]

[0062] The number of parallel channels used is N = 32 (determined by flow field uniformity simulation);

[0063] S3, curvature radius gradient design: arrange the curvature radius of the arc-shaped flow channel 8 according to an exponential law, and construct the flow resistance differential:

[0064]

[0065] Through ANSYS Fluent multi-physical field coupling simulation verification, the flow velocity fluctuation coefficient can be controlled within ±2.1% (CV value), and the outlet pulsatile pressure amplitude measured by the micro pressure sensor reaches 8.3kPa±0.5kPa, which proves that it can accurately reproduce the mechanical environment of capillary pulsation (p>0.05, ANOVA test).

[0066] The fluid buffer structure 5 adopts a tapering design, and the cross-sectional area of the flow channel follows an exponential decay law along the flow direction (x-axis direction):

[0067] A(x) = A0·e -βx (0≤χ≤L);

[0068] In the formula, A0 is the initial cross-sectional area at the inlet, β is the cross-sectional area decay coefficient optimized by CFD simulation, and L is the total length of the transition unit. The set design is solved by coupling with the Navier-Stokes equation, and the flow velocity field distribution equation is derived:

[0069]

[0070] Where v0 is the inlet flow velocity, and α is the flow velocity decay gradient coefficient, which is obtained by joint calibration of the rheological properties of red blood cell suspension (Casson fluid model, yield stress τ y = 0.5mPa) and the wall slip boundary condition (slip coefficient γ = 0.92). The smaller end of the fluid buffer structure 5 is in communication with the fatigue loading structure 4, and the larger end of the fluid buffer structure 5 is in communication with the bionic detection structure 6. In this embodiment, the fluid buffer structure satisfies the fluid velocity field regulation equation, realizes linear decay of flow velocity from 15mm / s to 0.4mm / s, and matches the physiological flow velocity 0.3-0.7mm / s in human capillaries.

[0071] The bionic detection structure 6 and the fatigue loading structure 4 are each provided with a plurality of micro-column arrays arranged orthogonally, the micro-column array in the fatigue loading structure has a size of 4 μm x 5 μm x 15 μm, and has 5100 columns along the flow direction, so as to ensure that the red blood cells flow in one direction to realize 5100 times of extrusion, and to cause cumulative fatigue damage to the red blood cells, and the cross-sectional size of 4 μm is used to increase the extrusion amplitude of the red blood cells, because the greater the extrusion and deformation of the red blood cells in the flow process, the more obvious the fatigue damage phenomenon, so as to further accelerate the in-vitro fatigue progress of the red blood cells and shorten the in-vitro experimental time. The length of 15 μm is used to reduce the total length of the flow channel of the pulsating fatigue loading area and the pressure at the inlet, because the longer the total flow channel, the greater the pressure at the inlet.

[0072] The micro-column array in the bionic detection structure 6 has a size of 5 μm x 5 μm x 30 μm, and has 10 columns along the flow direction, the size of 5 μm is used to simulate the size of the capillary in the body, and the tapered fluid buffer structure 5 is used to reduce the fluid velocity, so as to ensure that the cell flow rate of the red blood cells in the bionic detection structure 6 is consistent with the actual capillary flow rate in the body, the flow deformation of the fatigue red blood cells generated by the fatigue loading structure 4 in the bionic detection structure 6 can be observed, and the actual situation in the body can be directly reflected.

[0073] In the embodiment, the surface of the bionic detection structure 6 is provided with a periodically distributed nano-grating structure, the grating period is 200 nm ± 20 nm, and the depth is 50 nm ± 5 nm, which satisfies the light scattering enhancement condition:

[0074]

[0075] wherein λ = 500-600 nm is the imaging wavelength, η PDMS = 1.43 is the material refractive index, and Λ is the grating period, and the high-speed microscopic imaging system can realize the sub-pixel resolution (0.16 μm) of the red blood cell morphological parameters.

[0076] The method for using the integrated chip is as follows: red blood cells are injected into the chip, the red blood cells are guided to flow directionally in the chip by using a suction pump (a bidirectional injection pump is used in the embodiment), and the flow velocity, deformation index and shape relaxation time of the red blood cells are synchronously acquired by using a high-speed microscopic imaging system and an optical signal acquisition structure arranged outside the chip. The optical signal acquisition structure synchronously acquires the flow velocity, deformation index DI and shape relaxation time of the red blood cells. The optical signal acquisition structure comprises a dark field illumination module, a polarization analysis module and a synchronous control unit, and the dark field illumination module and the polarization analysis module adopt a coaxial spatial layout design. The dark field illumination module adopts a ring-shaped LED array, is integrated in a coaxial ring-shaped structure outside the objective lens of the high-speed microscopic imaging system, has a wavelength of 460nm±10nm and an incident angle of 60°±5°. The polarization analysis module comprises a rotatable analyzer (141, with an accuracy of 0.1°) and a four-quadrant photodetector, and is embedded in the rear end of the imaging light path and located at the sensor focal plane conjugate position of the high-speed camera. The synchronous control unit synchronously triggers the illumination and imaging at a frame frequency of 1000fps, and calculates the red blood cell velocity field distribution in real time based on the optical flow equation:

[0077] I x u+I y v+I t =0;

[0078] The deformation index (with an accuracy of ±0.05) and the shape relaxation time (with a measurement error of ≤5ms) are synchronously output.

[0079] Principles and effects of the application: the micro column array is constructed into a periodic optical enhancement structure through an orthogonal arrangement mode, when the ring-shaped light source of the dark field illumination module is irradiated at an inclination angle of 60°, the regular arrangement of the micro column array forms a multiple reflection-interference light path with the surface of the red blood cells, a Bragg diffraction enhancement effect is generated at a specific wavelength (460nm±10nm) to make the edge scattered light intensity of the red blood cells increased by 3-5 times, and a high-contrast dynamic image is provided for the high-speed microscopic imaging system; the optical signal acquisition structure realizes the extraction of the mechanical parameters. The red blood cell flow velocity is detected based on the sub-pixel level displacement (with an accuracy of 0.16um) of the red blood cell center in the continuous frame image, and the instantaneous flow rate is calculated in combination with the time interval; the deformation index DI is extracted, the maximum deformation variable in the long axis direction of the extruded red blood cell is extracted, and the deformation degree is calculated by comparing with the reference length in the relaxed state; the shape relaxation time is tracked, the time curve of the red blood cell from the extruded state to the original shape after passing through the micro column gap is tracked, and the membrane elasticity is quantified by using an exponential fitting model.

[0080] The application utilizes the high-frequency extrusion deformation of the red blood cell sample at the micron scale, realizes the fatigue accumulation effect of the red blood cell population, and then reduces the liquid flow rate by increasing the cross section to observe the flow extrusion deformation of the fatigue state red blood cells at the physiological flow rate, and the specific implementation steps are as follows:

[0081] S1, blood sample collection: using a sterile syringe and a disposable safety blood needle to extract 2 milliliters of venous blood sample from the arm of a healthy volunteer; the extracted blood sample is injected into a vacuum blood collection tube containing EDTA anticoagulant (purple); low temperature storage (2-8℃ constant temperature storage box); record the date and time of blood collection; according to the biological safety standard of blood sample, the collected blood sample is properly stored; and the blood sample is sent to the laboratory for further analysis and detection.

[0082] S2, red blood cell suspension preparation: the collected whole blood sample is transferred to a centrifuge tube and diluted with PBS solution; the diluted whole blood solution is washed three times using a centrifuge pump, each time at 4℃, 400xg for 5min, and the supernatant is carefully removed using a pipette, leaving the red blood cell precipitate; the washed red blood cells are resuspended in PBS solution for dilution to maintain the cell survival environment.

[0083] S3, red blood cell suspension preparation: the collected whole blood sample is transferred to a centrifuge tube and diluted with PBS solution; the diluted whole blood solution is washed three times using a centrifuge pump, each time at 4℃, 400xg for 5min, and the supernatant is carefully removed using a pipette, leaving the red blood cell precipitate; the washed red blood cells are resuspended in PBS solution for dilution; the prepared red blood cell suspension is stored at 4℃ for standby.

[0084] S4, red blood cell fatigue experiment: using a 500ul injection pump to inject the prepared red blood cell suspension into the chip at a flux of 0.4ul / min, according to the experimental design, the red blood cells complete 5100 times of flow cycle in the chip, which takes about 2h, and the fatigued red blood cell sample flows out of the chip and enters the reverse injector, and the fatigued red blood cell sample is collected for subsequent experimental exploration.

[0085] S5, observation of flow deformation of fatigued red blood cells: high-speed camera is used to obtain the flow deformation pattern of red blood cells in the observation area; existing image processing methods such as gray scale conversion and edge detection are used to pretreat the red blood cell pattern, and the boundary and morphology of red blood cells are segmented out, and gray scale stretching is carried out based on CLAHE algorithm (contrast limited adaptive histogram equalization), the window size is set to 32x32 pixels, and the contrast threshold limit is 2.0, to enhance the contrast of red blood cells and background (ΔE≥15), edge detection uses Canny operator for multi-scale edge detection, and the long axis length L max of red blood cells before deformation is extracted and measured; the long axis length L max extracted above is used to calculate the elongation ratio of red blood cells before and after deformation. In this embodiment, according to the red blood cell deformation index formula: DI=L maxL0, calculate the deformation index of red blood cells; quantify the degree of deformation of red blood cells, and represent the deformation performance of red blood cells.

[0086] S6, fatigue state red blood cell shape proportion observation: obtain the red blood cell sample after fatigue from the reverse syringe, add the fatigue red blood cell sample into glutaraldehyde solution, and fix it at 4°C for 1h; use a pipette to suck the fixed red blood cell suspension, drop it on a clean glass slide covered with poly-l-lysine film, and store it in a humid environment for 30min to allow the red blood cells to naturally sediment and adsorb; immerse the adsorbed glass slide in different concentrations of alcohol solution in turn; use liquid carbon dioxide to perform critical point drying on the broken pieces in a vacuum device; use double-sided tape to paste the dried broken pieces on a sample stage, spray gold plating film, and use a scanning electron microscope to observe different red blood cell shapes to prepare scanning electron microscope images of red blood cells.

[0087] Result analysis and evaluation:

[0088] 1. Fatigue damage effect:

[0089] As shown in Figure 7 , after 5100 cycles of extrusion, the red blood cell population showed significant morphological differentiation: normal biconcave disc-shaped cells (65.2% ± 3.1%), spiny cells (24.7% ± 2.8%), and spherical cells (10.1% ± 1.9%), indicating that red blood cells undergo morphological evolution during mechanical fatigue, thereby confirming that mechanical fatigue triggers cell membrane remodeling.

[0090] 2. Flow velocity characterization:

[0091] As shown in Figure 6 , Figure 8 , and Figure 9 , during the flow process, normal red blood cells flowed in a slipper shape in the microchannel (DI = 1.31 ± 0.08), and completed migration through a folding extrusion mechanism, with an average flow rate of 0.32 ± 0.05 mm / s; while spiny cells (DI = 1.18 ± 0.07) and spherical cells (DI = 1.09 ± 0.03) were delayed in flow due to increased membrane rigidity (flow rates decreased by 15.6% and 67.8%, respectively); channel retention was observed in some spherical cells, confirming the loss of mechanical homogeneity. These results show that cumulative damage triggers a catastrophic degradation of cell mechanical properties.

[0092] The experimental results systematically reveal the fatigue damage dynamics of red blood cells under cyclic stress, verify the technical advantages of the chip in high-throughput analysis of cell mechanical phenotypes (throughput 104cells / min) and pathological threshold determination, and provide a quantifiable diagnostic benchmark (CV = 3.2%, meeting the ISO 13485 standard) for clinical evaluation of red blood cell mechanical properties.

[0093] The application realizes the functional coupling of red blood cell fatigue loading and dynamic observation under in-vitro conditions, can effectively, non-destructively and quickly measure the flow deformation characteristics of fatigue-state red blood cells, and has the characteristics of high-throughput, non-contact cell mechanics performance detection, meanwhile, the system has low experience requirement for operators, and has high experimental stability and good repeatability.

[0094] As shown in Figure 10 The processing method of the red blood cell mechanical fatigue damage and mechanical characterization integrated chip based on multi-stage flow field regulation of the application is as follows:

[0095] Step one: uniformly coating a photoresist layer on the surface of a silicon substrate by using a spin coating process;

[0096] Step two: covering the silicon substrate with a mask plate of the flow channel structure of the integrated chip, and performing ultraviolet exposure;

[0097] Step three: forming a channel mold through post-baking treatment, chemical development and high-temperature curing process;

[0098] Step four: pouring PDMS (10:1) containing a curing agent on the surface of the mold, removing the base mold after high-temperature curing forming, and cutting and punching the demolded PDMS;

[0099] Step five: using oxygen plasma surface treatment technology to bond the PDMS flow channel layer and the glass substrate 1, and finally complete the chip packaging.

[0100] The above is only an embodiment of the application, and the common knowledge of specific structures and / or characteristics in the scheme is not described in detail. It should be pointed out that for those skilled in the art, without departing from the structure of the application, some modifications and improvements can be made, which should be regarded as the protection scope of the application, and these will not affect the effect and practicality of the application. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. An integrated chip for red blood cell mechanical fatigue damage and mechanical characterization, characterized by: The invention comprises a substrate on which a PDMS functional layer is provided; the substrate and the PDMS functional layer are tightly bonded to form a composite microchannel system with bionic flow field characteristics, wherein the composite microchannel system comprises a fatigue loading structure, a fluid buffer structure and a bionic detection structure connected in sequence, wherein the fatigue loading structure comprises a straight microchannel and an arc-shaped flow channel connected alternately, wherein the fluid buffer structure adopts a tapered design, wherein the smaller end of the fluid buffer structure is connected to the fatigue loading structure, and the larger end of the fluid buffer structure is connected to the bionic detection structure, and wherein a plurality of orthogonally arranged microcolumn arrays are provided in both the bionic detection structure and the fatigue loading structure.

2. The integrated chip for red blood cell mechanical fatigue damage and mechanical characterization according to claim 1, characterized in that: The size of the micro-column array in the fatigue loading structure is 4 μm×5 μm×15 μm, and the size of the micro-column array in the bionic detection structure is 5 μm×5 μm×30 μm.

3. The integrated chip for red blood cell mechanical fatigue damage and mechanical characterization according to claim 1, characterized in that: The flow channel design of the fatigue loading structure follows the modified Womersley pulsating flow model, and its pressure gradient equation is expressed as: Where: Q = 1.2 × 10 -3 Pa·s is the viscosity of PBS solution at 37°C, Q = 0.4 μL / min is the volume flow rate, corresponding to an inlet flow rate of 15 mm / s, and ρ = 1030 kg / m 3 , is the density of red blood cell suspension, and a gradient flow resistance network is constructed by alternating straight microchannel arrays and curved flow channels.

4. The integrated chip for red blood cell mechanical fatigue damage and mechanical characterization according to claim 3, characterized in that: The inlet flow velocity design method of the fatigue loading structure is as follows: S1. Optimization of inlet flow resistance: A progressively contracting inlet was used, and the contraction angle was determined to be 5° through numerical simulation of the Navier-Stokes equation, which reduced the inlet pressure loss to 0.8 kPa. S2. Fluidic parameters of straight microchannel array: Flow resistance of single straight channel: The number of parallel channels used is: N = 32, determined by flow field uniformity simulation; S3. Curvature radius gradient design: Arrange the curvature radius of the arc flow channel according to the exponential law and construct the flow resistance differential: Verified by ANSYS Fluent multi-physics field coupling simulation, this design can control the flow velocity fluctuation coefficient within ±2.1%, and the outlet pulsating pressure amplitude measured by the micro-pressure sensor reaches 8.3kPa±0.5kPa, proving that it can accurately reproduce the mechanical environment of capillary pulsation.

5. The integrated chip for red blood cell mechanical fatigue damage and mechanical characterization according to claim 1, characterized in that: The cross-sectional area of ​​the flow channel of the fluid buffer structure follows an exponential decay law along the flow direction: A(x)=A0·e -βx (0≤X≤L); Where A0 is the initial cross-sectional area of ​​the inlet, β is the cross-sectional area attenuation coefficient optimized by CFD simulation, and L is the total length of the transition unit. By coupling with the Navier-Stokes equation, the velocity field distribution equation is derived: Where v0 is the inlet flow velocity and α is the velocity attenuation gradient coefficient, whose value is obtained by jointly calibrating the rheological properties of the red blood cell suspension and the wall slip boundary condition.

6. An integrated chip for mechanical fatigue damage and mechanical characterization of red blood cells according to any one of claims 1 to 5, characterized in that: The integrated chip is used as follows: red blood cells are injected into the chip, a suction pump is used to guide the red blood cells to flow in a directional manner within the chip, and the red blood cell flow velocity, deformation index, and morphological relaxation time are synchronously obtained through a high-speed microscopic imaging system and an optical signal acquisition structure arranged outside the chip.

7. The integrated chip for red blood cell mechanical fatigue damage and mechanical characterization according to claim 3, characterized in that: The surface of the bionic detection structure is provided with a periodically distributed nano-grating structure with a grating period of 200nm±20nm and a depth of 50nm±5nm, which meets the light scattering enhancement conditions: Where λ = 500-600nm is the imaging wavelength, η POMS =1.43 is the refractive index of the material, and Λ is the grating period.

8. The integrated chip for red blood cell mechanical fatigue damage and mechanical characterization according to claim 7, characterized in that: The optical signal acquisition structure includes a dark-field illumination module, a polarization analysis module, and a synchronization control unit. The dark-field illumination module and the polarization analysis module are designed with a coaxial spatial layout. The specific method is as follows: the dark-field illumination module is integrated into the periphery of the objective lens of the high-speed microscopy imaging system in a coaxial ring structure. Its ring-shaped LED array and the objective lens form a 60°±5° inclined illumination light path; the polarization analysis module includes a rotatable polarizer and a four-quadrant photodetector, and is embedded in the rear end of the imaging light path, located at a conjugate position of the sensor focal plane of the high-speed camera; the synchronization control unit synchronously triggers illumination and imaging at a frame rate of 1000 fps, based on the optical flow equation: I x u+I y v+I t =0; Calculate the red blood cell velocity field distribution and synchronously output the deformation index and morphological relaxation time.

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