Microfluidic detection system and method for erythrocytes of diabetic patient

Abstract

The invention relates to the technical field of cell detection and analysis, and particularly discloses a microfluidic detection system and method for erythrocytes of diabetic patients. The system comprises a PDMS micro-fluidic chip, an equal-diameter bionic micro-channel array, a closed-loop constant-pressure pump and a high-speed imaging module. Synchronous observation of dynamic flow deformation behaviors of red blood cells is realized by simulating a diabetes microvessel shearing environment and combining dark field illumination and a Canny edge detection algorithm. The method comprises the steps of blood sample anticoagulation treatment, erythrocyte suspension gradient centrifugal purification, dynamic flow deformation analysis under pulsating pressure driving and scanning electron microscope morphological characterization. The system breaks through the limitation of the traditional technology, can effectively, losslessly and rapidly measure the flow deformation characteristics of erythrocytes of diabetic patients under different glycosylated hemoglobin concentrations, has high-throughput and non-contact cell mechanical property detection characteristics, has low experience requirements on operators, and has high stability and repeatability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell detection analysis, in particular to a microfluidic detection system and method for red blood cells of diabetic patients. BACKGROUND

[0002] Diabetes is the most common chronic metabolic disease in China. Microcirculation disorder caused by diabetes is the pathological basis of diabetic pan-angiopathy, which involves target organs such as cardiovascular system, brain and blood vessels, retina, kidney and peripheral nervous system. Abnormal rheological properties of red blood cells are an important pathological link leading to microcirculation disorder and vascular complications. Red blood cell morphology mainly includes cell structure and shape. Under the condition of long-term high blood glucose, the double-concave disc structure of red blood cells will gradually be lost, and the shape will be elongated, the volume will be reduced, the rigidity index will be increased, and the deformability will be reduced. These changes affect the rheology of blood and the transport function of red blood cells, making it difficult for red blood cells to pass through small blood vessels and causing local tissue ischemia and hypoxia, thereby increasing the risk of diabetic microvascular complications.

[0003] Long-term high blood glucose environment may cause remodeling of red blood cell membrane structure. Na + -K + -ATPase is an important membrane-bound enzyme. Long-term high blood glucose environment inhibits its activity, causes intracellular sodium retention and osmotic pressure imbalance, and leads to compensatory swelling of red blood cells and volume increase. Inhibition of Ca 2+ -Mg 2+ -ATPase activity causes intracellular calcium overload, which destroys the stability of the membrane skeleton protein network through phosphorylation, and promotes the increase of spiculated red blood cells.

[0004] Traditional blood rheology detectors can measure blood viscosity, aggregation index and other macroscopic parameters, but cannot observe the dynamic deformation process of red blood cells in the biomimetic microchannel. In recent years, microfluidic technology has been applied to cell mechanics research, but existing schemes still have problems such as multi-parameter detection discretization, dynamic tracking inaccuracy, and insufficient pathological model adaptability, which cannot meet the needs of precise evaluation of red blood cells of diabetic patients. SUMMARY

[0005] 1. Technical problem to be solved

[0006] The purpose of the present application is to solve the problem that traditional blood rheology analyzers can only measure single parameters such as whole blood viscosity and aggregation index, and lack comprehensive evaluation of the correlation between the dynamic deformation process of red blood cells and the concentration gradient of glycated hemoglobin. The present application provides a microfluidic detection system for detecting red blood cell dynamics analysis and morphology characterization of diabetic patients.

[0007] 2. Technical solution

[0008] The application discloses a microfluidic detection system for red blood cells of a diabetic patient, which comprises a PDMS microfluidic chip and a constant pressure pump, the constant pressure pump is used for pumping red blood cells of the diabetic patient into the PDMS microfluidic chip, the PDMS microfluidic chip is internally provided with a microchannel array structure, the microchannel array structure is composed of a plurality of equidiameter biomimetic microchannels arranged side by side, the equidiameter biomimetic microchannels have a cross-sectional dimension of 5mu m*5mu m, and the surface is subjected to oxygen plasma modification treatment; the constant pressure pump generates a 5-20mbar pulsating pressure field through a closed-loop feedback algorithm; the microchannel array structure is externally integrated with a high-speed camera and a microscope; and the microscope and the high-speed camera are jointly used for detecting the morphological change of the red blood cells in the microchannel array structure.

[0009] Further, the flow channel length of the microchannel array structure is 500mu m, the channel inner wall roughness is less than or equal to 50nm, the flow resistance gradient is optimized through a Navier-Stokes equation, and a shear rate of 40s -1 .

[0010] Further, the constant pressure pump is internally provided with a PID control module, the pressure loading frequency is 1-5Hz, and the response time is less than or equal to 10ms.

[0011] Further, the microscope is provided with a dark field illumination module, the light source wavelength of the dark field illumination module is 532nm+ / -5nm, and the adjustment range of the light intensity is 10-100mW / cm 2 .

[0012] Further, the integrated detection system further comprises an intelligent analysis unit, the intelligent analysis unit comprises:

[0013] a red blood cell extraction module, the red blood cell extraction module adopts a Canny edge detection algorithm to extract the red blood cell contour from the video and the picture acquired by the microscope and the high-speed camera;

[0014] a velocity field reconstruction module, the velocity field reconstruction module calculates the red blood cell flow velocity through a formula .

[0015] a morphological characterization module, the morphological characterization module calculates the red blood cell stretch ratio through a stretch ratio formula .

[0016] Further, the detection method of the integrated detection system comprises the following steps:

[0017] S1, blood sample collection and treatment: 2ml of venous blood sample is extracted from the arm of a diabetic patient; the extracted blood sample is injected into a vacuum blood collection tube containing an EDTA anticoagulant, and is stored at low temperature; the date, time and glycated hemoglobin concentration of blood collection are recorded; the blood sample is sent to a laboratory for analysis and detection according to the blood sample biological safety specification;

[0018] S2, Preparation of red blood cell suspension: The collected whole blood sample was transferred to a centrifuge tube and diluted with PBS solution; the diluted whole blood solution was washed three times using a centrifugal pump, each time at 4°C, 400xg for 5 min, and the supernatant was carefully removed using a pipette gun, leaving a red blood cell precipitate; the washed red blood cells were resuspended in a PBS solution for dilution, and the prepared red blood cell suspension was stored at 4°C for standby use;

[0019] S3, Dynamic rheological analysis: the prepared red blood cell suspension was driven into the chip using a constant pressure pump at different pressures, and the red blood cells were deformed by flow extrusion in the array structure of the restricted microchannels in the PDMS microfluidic chip according to the experimental design; a high-speed camera was used to obtain the flow deformation pattern of the red blood cells in the observation area; the image was preprocessed by gray scale conversion and edge detection to segment the boundary and morphology of the red blood cells; the contour and morphological features of the red blood cells were obtained through image processing technology; the flow of the red blood cells was observed and recorded using computer vision methods; the initial length D0 of the red blood cells and the time T0 when the red blood cells completely entered the equal-diameter microchannels were obtained; the time T1 when the red blood cells were about to leave the equal-diameter microchannels and the length D(t) of the red blood cells after stretching in the microchannels were obtained; the flow distance L of the red blood cells in the time period was extracted and measured; the flow velocity of the red blood cells in the biomimetic microchannels was calculated using T0, T1 and L, and the specific formula is as follows:

[0020]

[0021] The initial length D0 of the red blood cells and the length D(t) of the red blood cells after stretching in the microchannels were calculated to obtain the stretching ratio of the red blood cells after entering the microchannel array structure, and the formula is as follows:

[0022]

[0023] S4, Morphological characterization: Red blood cell morphology shooting: the red blood cell sample was added to a glutaraldehyde solution and fixed at room temperature for 1 h; the fixed red blood cell suspension was taken with a pipette gun and dropped on a clean glass slide covered with a poly-L-lysine film, and the red blood cells were naturally precipitated and adsorbed in a humid environment for 30 min; the adsorbed glass slide was sequentially soaked in different concentrations of alcohol solution; the glass slide was critical point dried with liquid carbon dioxide in a vacuum device; the dried glass slide was pasted on a sample stage and gold-plated, and different red blood cell morphologies were observed using a scanning electron microscope to prepare scanning electron microscope images of red blood cells and compare the relative proportions of normal biconcave disc-shaped red blood cells and other atypical red blood cells at different glycated hemoglobin concentrations.

[0024] Further, the anti-coagulant optimization scheme in step S1 is: EDTA concentration is 1.2 mg / mL ± 0.1 mg / mL, and the storage time is ≤6 h.

[0025] Further, the scanning electron microscope sample processing in step S4 includes three groups of time nodes: non-fixing processing, processing immediately after fixing, and processing after 24 h of 4℃ storage, for evaluating morphological stability.

[0026] Compared with the prior art, the beneficial effects of the scheme are:

[0027] The present application realizes the functional coupling of the in-vitro flow and dynamic observation of the red blood cells of diabetic patients, can effectively, non-destructively and quickly measure the flow deformation characteristics of the red blood cells of diabetic patients, and has the characteristics of high-throughput, non-contact cell mechanics performance detection, and the experience requirement of the operator is not high, and the system has high experimental stability and good repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A microfluidic detection system flow process diagram for red blood cells of diabetic patients is provided in the present application;

[0029] Figure 2 A red blood cell flow deformation diagram is taken in the experiment of the red blood cells passing through the micro-channel array structure in the present embodiment;

[0030] Figure 3 A flow velocity analysis diagram of the red blood cells of diabetic patients in the micro-channel is provided in the present application;

[0031] Figure 4 A stretch ratio analysis diagram of the red blood cells of diabetic patients after deformation and stretching in the micro-channel is provided in the present application;

[0032] Figure 5 A scanning electron microscope morphological characterization of the red blood cells of diabetic patients is provided in the present application. DETAILED DESCRIPTION

[0033] The present application will be further described in detail through specific embodiments:

[0034] EMBODIMENT

[0035] As shown in the drawings, Figures 1 to 5 A microfluidic detection system for red blood cells of diabetic patients includes a micro-channel array structure, a PDMS microfluidic chip, and a constant pressure pump. The PDMS microfluidic chip is connected to the constant pressure pump through a microfluidic conduit. The constant pressure pump is provided with a PID control module. The pressure loading frequency is controlled at 1-5 Hz through the PID control module, the response time is ≤10 ms, and the constant pressure pump generates a 5-20 mbar pulsatile pressure field through a closed-loop feedback algorithm. The micro-channel array structure is composed of a plurality of equal-diameter biomimetic micro-channels arranged side by side.Figure 1 The cross-sectional size of the isodiametric biomimetic microchannel is 5 μm x 5 μm, the surface is treated by oxygen plasma modification, and the contact angle is ≤30°; at the same time, the flow channel length of the microchannel array structure is 500 μm, the inner wall roughness of the channel is ≤50 nm, the flow resistance gradient is optimized by the Navier-Stokes equation, and a shear rate of 40 s -1 The outside of the microchannel array structure is integrated with a high-speed camera and a microscope. The high-speed camera is a Hamamatsu Orca-flash 2.8 with a frame rate ≥800 fps, and the microscope is composed of a 40x oil lens and a 10x eyepiece. The microscope and the high-speed camera are used together to detect the morphological changes of red blood cells in the microchannel array structure, with a spatial resolution of 0.16 μm. The microscope is equipped with a dark field illumination module, and the light source wavelength of the dark field illumination module is 532 nm±5 nm, and the light intensity adjustment range is 10-100 mW / cm 2 .

[0036] The detection integrated system also includes an intelligent analysis unit, which includes:

[0037] A red blood cell extraction module extracts the outline of red blood cells in the red blood cell video and picture obtained by the microscope and the high-speed camera by using a Canny edge detection algorithm;

[0038] A velocity field reconstruction module calculates the flow velocity of red blood cells by the formula with an accuracy of ±10 μm / s;

[0039] A morphological characterization module calculates the stretch ratio of red blood cells by the formula .

[0040] The detection method of the detection integrated system of the embodiment includes the following steps:

[0041] S1, blood sample collection and processing: 2 milliliters of venous blood sample is extracted from the arm of a diabetic patient by using a sterile syringe and a disposable safety blood collection needle; the extracted blood sample is injected into a vacuum blood collection tube containing EDTA anticoagulant, and stored at low temperature (2-8℃ constant temperature storage box); the date, time and glycosylated hemoglobin concentration of blood collection are recorded; the collected blood sample is properly stored according to the biological safety standard of blood sample; and the blood sample is sent to the laboratory for analysis and detection. In this step, the EDTA concentration is 1.2 mg / mL±0.1 mg / mL, and the storage time is ≤6 h.

[0042] S2, preparation of red blood cell suspension: the collected whole blood sample was transferred to a centrifuge tube and diluted with PBS solution; the diluted whole blood solution was washed three times using a centrifugal pump, each time at 4°C, 400xg for 5min, and the supernatant was carefully removed using a pipette gun, leaving the red blood cell precipitate; the washed red blood cells were resuspended in PBS solution for dilution, and the prepared red blood cell suspension was stored at 4°C for standby.

[0043] S3, dynamic rheological analysis: the prepared red blood cell suspension was driven into the inside of the chip using a constant pressure pump at different pressures (10mbar, 15mbar, 20mbar), and the red blood cells were deformed by flow extrusion in the restricted microchannel region of the PDMS microfluidic chip according to the experimental design; a high-speed camera was used to obtain the flow deformation pattern of the red blood cells in the observation region, and existing image processing methods such as gray scale conversion and edge detection were used to pretreat the flow deformation pattern of the red blood cells, so as to facilitate the segmentation of the boundary and morphology of the red blood cells, and to obtain the contour and morphological features of the red blood cells; existing computer vision methods were used to observe and record the flow of the red blood cells (such as Figure 2 The length D0 of the red blood cells at the initial moment and the moment T0 when the red blood cells completely entered the constant-diameter microchannel were obtained; the moment T1 when the red blood cells were about to leave the constant-diameter microchannel and the length D(t) of the red blood cells after stretching in the microchannel were obtained; the flow distance L of the red blood cells in the time period was extracted and measured; the flow velocity of the red blood cells in the biomimetic microchannel was calculated using T0, T1 and L, and the specific formula was as follows:

[0044]

[0045] In this embodiment, statistical analysis was performed on the above data, the average value of the red blood cell flow rate of patients with different glycated hemoglobin concentrations was calculated, and the trend of the change of the red blood cell flow rate was evaluated (such as Figure 3 ).

[0046] The initial length D0 of the red blood cells and the length D(t) of the red blood cells after stretching in the microchannel were used to calculate the stretching ratio of the red blood cells after entering the microchannel, and the formula was as follows:

[0047]

[0048] In this embodiment, statistical analysis was performed on the above data, the mean, maximum, minimum, variance and standard deviation parameters were calculated, and the trend of the change of the red blood cell stretching ratio under different glycated hemoglobin concentrations was evaluated (such as Figure 4 ).

[0049] S4, morphological characterization: red blood cell morphology shooting: add the red blood cell sample into glutaraldehyde solution, fix at room temperature for 1 h; use a pipette to suck the fixed red blood cell suspension, drop on a clean glass slide covered with poly-L-lysine film, and store in a humid environment for 30 min, let the red blood cells naturally sediment and adsorb; immerse the adsorbed glass slide in different concentrations of alcohol solution in turn; dry the glass slide in a vacuum device with liquid carbon dioxide at the critical point; use double-sided tape to stick the dried glass slide on the sample table and spray gold film, use scanning electron microscope to observe different red blood cell morphology, prepare red blood cell scanning electron microscope image (such as Figure 5 ), compare the relative proportion of normal biconcave disc-shaped red blood cells and other atypical red blood cells such as acanthocytes under different glycated hemoglobin concentrations. In this step, the scanning electron microscope sample processing includes three groups of time nodes: no fixation treatment, immediate treatment after fixation, and treatment after 24 h storage at 4℃, which is used to evaluate the morphological stability.

[0050] The embodiment constructs a "pressure loading-dynamic observation-morphological characterization" trinity detection system, realizes the synchronous and accurate analysis of red blood cell dynamic deformation behavior and morphological heterogeneity under in vitro conditions, can effectively, non-destructively and quickly measure the flow deformation characteristics of red blood cells under diabetic conditions, and has high throughput, non-contact cell mechanics performance detection characteristics, at the same time, the system has low experience requirement for operators, has high experimental stability and good repeatability.

[0051] The above is only an embodiment of the present 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 present application, a number of modifications and improvements can be made, which should be regarded as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in this application should be subject to the content of its claims, and the specific implementation mode and the like recorded in the specification can be used to explain the content of the claims.

Claims

1. A microfluidic detection system for red blood cells of diabetic patients, characterized by: The device comprises a PDMS microfluidic chip and a constant pressure pump, wherein the constant pressure pump is used to pump red blood cells of diabetic patients into the PDMS microfluidic chip. The PDMS microfluidic chip is provided with a microchannel array structure, which is composed of a plurality of parallel bionic microchannels of equal diameter, each having a cross-sectional size of 5 μm × 5 μm and a surface modified by oxygen plasma. The constant pressure pump generates a 5-20 mbar pulsating pressure field through a closed-loop feedback algorithm; a high-speed camera and a microscope are integrated on the outside of the microchannel array structure; the microscope and the high-speed camera are used together to detect changes in red blood cell morphology within the microchannel array structure.

2. A microfluidic detection system for red blood cells of diabetic patients according to claim 1, characterized in that: The flow channel length of the microchannel array structure is 500 μm, the channel inner wall roughness is ≤ 50 nm, and the flow resistance gradient is optimized by the Navier-Stokes equation, generating a shear rate of 40 s at an inlet pressure of 15 mbar. -1 .

3. The microfluidic detection system for red blood cells of diabetic patients according to claim 2, characterized in that: The constant pressure pump is provided with a PID control module, the pressure loading frequency is 1-5 Hz, and the response time is ≤10 ms.

4. The microfluidic detection system for red blood cells of diabetic patients according to claim 1, characterized in that: The microscope is equipped with a dark field illumination module. The light source wavelength of the dark field illumination module is 532nm±5nm, and the light intensity adjustment range is 10-100mW / cm 2 .

5. The microfluidic detection system for red blood cells of diabetic patients according to claim 1, characterized in that: It also includes an intelligent analysis unit, which includes: A red blood cell extraction module, which uses the Canny edge detection algorithm to extract red blood cell contours from videos and images acquired by a microscope and a high-speed camera; The velocity field reconstruction module is constructed by the formula Calculate red blood cell velocity; The morphological characterization module is characterized by the stretching ratio formula Calculate the red blood cell stretch ratio.

6. A microfluidic detection system for red blood cells of diabetic patients according to any one of claims 1 to 5, characterized in that: The detection method of the integrated detection system comprises the following steps: S1. Blood sample collection and processing: Draw 2 ml of venous blood sample from the diabetic patient's arm; inject the drawn blood sample into a vacuum tube containing EDTA anticoagulant and store at low temperature; record the date, time, and glycated hemoglobin concentration of blood collection; and send the blood sample to the laboratory for analysis and testing in accordance with blood sample biosafety regulations; S2. Preparation of red blood cell suspension: Transfer the collected whole blood sample to a centrifuge tube and dilute with PBS solution. Wash the diluted whole blood solution three times using a centrifugal pump, centrifuging each time at 4°C and 400×g for 5 min. Carefully remove the supernatant using a pipette, leaving the red blood cell pellet. Resuspend the washed red blood cells in PBS solution and dilute. Store the prepared red blood cell suspension at 4°C until ready for use. S3. Dynamic rheological analysis: A constant pressure pump is used to drive the prepared red blood cell suspension into the chip at different pressures. According to the experimental design, the red blood cells produce flow extrusion deformation in the confined microchannel array structure in the PDMS microfluidic chip; a high-speed camera is used to obtain the flow deformation graph of the red blood cells in the observation area; grayscale conversion and edge detection are used to preprocess the image to segment the boundaries and morphology of the red blood cells; the outline and morphological characteristics of the red blood cells are obtained through image processing technology; computer vision methods are used to observe and record the flow of red blood cells; the length D0 of the red blood cells at the initial moment and the time T0 when the red blood cells completely enter the equal-diameter microchannel are obtained; the time T1 when the red blood cells are about to leave the equal-diameter microchannel and the length D(t) of the red blood cells after being stretched in the microchannel are obtained; the flow distance L of the red blood cells in the time period is extracted and measured; the flow velocity of the red blood cells in the bionic microchannel is calculated using T0, T1 and L. The specific formula is as follows: The stretching ratio of the red blood cells after entering the microchannel array structure is calculated using the extracted initial length D0 of the red blood cells and the length D(t) of the red blood cells after stretching in the microchannel. The formula is as follows: S4. Morphological characterization: Red blood cell morphology photography: Add the red blood cell sample to glutaraldehyde solution and fix it at room temperature for 1 hour; use a pipette to draw up the fixed red blood cell suspension and drop it on a clean glass slide covered with a poly-L-lysine membrane, and store it in a humid environment for 30 minutes to allow the red blood cells to naturally precipitate and adsorb; soak the adsorbed glass slide in alcohol solutions of different concentrations in turn; perform critical point drying of the glass slide with liquid carbon dioxide in a vacuum device; stick the dried glass slide on the sample stage and spray gold coating, use a scanning electron microscope to observe different red blood cell morphologies, prepare red blood cell scanning electron microscope images, and compare the relative proportions of normal biconcave disc-shaped red blood cells and other abnormal red blood cells such as spinous-shaped red blood cells at different glycated hemoglobin concentrations.

7. The microfluidic detection system for red blood cells of diabetic patients according to claim 6, characterized in that: In step S1, an optimized anticoagulant solution was used: EDTA concentration was 1.2 mg / mL ± 0.1 mg / mL, and storage time was ≤ 6 h.

8. The microfluidic detection system for red blood cells of diabetic patients according to claim 6, characterized in that: The SEM sample processing in step S4 includes three time points: unfixed processing, processing immediately after fixation, and processing after storage at 4°C for 24 h, which is used to evaluate morphological stability.

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