CN108855256B - Microfluidic chip and method for detecting deformability of red blood cells - Google Patents
Microfluidic chip and method for detecting deformability of red blood cells Download PDFInfo
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Abstract
The invention discloses a micro-fluidic chip for detecting erythrocyte deformability and a method thereof. The invention adopts a wavy microfluidic channel which comprises an inlet, a first buffer straight channel, a first connecting part, a wave channel, a second connecting part, a second buffer straight channel and an outlet which are connected in sequence, and when continuous extrusion stress is provided, the red blood cells are bent and deformed along with the change of waves, so that stress in another form is provided; injecting a perfusion liquid into a micro-fluidic chip for detecting the deformability of the red blood cells by adopting a perfusion liquid propelling device and a pressure gradient method controlled by a height difference, and propelling the flow of the perfusion liquid in the micro-fluidic chip; the invention can achieve the purpose of detecting the deformability of the red blood cells in vitro, can effectively simulate the blood microcirculation environment of human capillaries from the diameter and the shape, and particularly can simulate the extrusion condition of the red blood cells at the spleen in vitro experiment.
Description
Technical Field
The invention relates to a microfluidic chip technology, in particular to a microfluidic chip for detecting erythrocyte deformability and a method thereof.
Background
The red blood cells are the blood cells with the largest quantity in blood, are in a double-sided concave round cake shape, have the diameter of 6-8 mu m, the thickness edge of about 2 mu m and the center of about 1 mu m, have extremely strong deformability, can pass through capillaries with the diameters smaller than the diameters for many times in the service life of 120 days without being damaged, and the deformability is the guarantee for maintaining various life activities such as blood circulation, material exchange, energy transfer and the like in a human body. A micro-fluidic system based on a micro-fluidic chip is a common method for detecting the deformability of red blood cells, and most of channels simulating that red blood cells extrude and pass through capillaries in the existing micro-fluidic chip are single-channel or multi-channel straight channels, so that extrusion stress perpendicular to the advancing direction is provided for the red blood cells passing through the channels to enable the red blood cells to deform so as to detect the deformability. However, the capillary vessels in human bodies have various winding and bending shapes, and the red blood cells are not only extruded by the vessel wall in the advancing process, but also bent and deformed in the continuous turning process, so that the traditional straight channel can only provide single extrusion stress for the red blood cells passing through the straight channel, and the simulation effect on the blood vessels is not good enough.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a micro-fluidic chip for detecting the deformability of red blood cells and a method thereof.
One objective of the present invention is to provide a microfluidic chip for detecting deformability of erythrocytes.
The micro-fluidic chip for detecting the deformability of the red blood cells comprises: the device comprises a substrate, a wave-shaped micro-flow channel and a cover plate; wherein, a positive pattern with a wavy micro-flow channel is formed on the hard template; the stock solution of the substrate is paved on the template to cover the whole male pattern, the base plate is formed after the stock solution is solidified, and the female pattern of the wavy micro-flow channel is formed on the surface of the base plate; the surface of the substrate with the negative pattern and the transparent cover plate are sealed together in a bonding mode, so that a closed channel, namely a wave-shaped micro-flow channel, is formed between the surface of the substrate and the cover plate; the wavy microfluidic channel comprises an inlet, a first buffer straight channel, a first connecting part, a wavy channel, a second connecting part, a second buffer straight channel and an outlet which are connected in sequence, the width of the first buffer straight channel and the width of the second buffer straight channel are larger than the maximum diameter of red blood cells, the height of the first buffer straight channel and the height of the second buffer straight channel are larger than the maximum thickness of the red blood cells, so that the red blood cells are not extruded when passing through, and the first connecting part and the second connecting part are isosceles trapezoids and are larger than the maximum thickness of the red blood cells; the width of the front end of the first connecting part is the same as that of the first buffering direct current channel, the width of the tail end of the first connecting part is the same as that of the wave channel, the width of the front end of the second connecting part is the same as that of the wave channel, and the width of the tail end of the second connecting part is the same as that of the second buffering direct current channel; the width of the wave channel is more than 1 μm and less than the minimum diameter of the red blood cells, and the height of the wave channel is more than 1 μm and less than the minimum diameter of the red blood cells, so that the red blood cells are extruded and deformed when passing through the wave channel, and the wave channel is in a curved shape; an inlet through hole and an outlet through hole are respectively formed in the substrate, the inlet through hole is communicated to an inlet of the wavy micro-flow channel, and the outlet through hole is communicated to an outlet of the wavy micro-flow channel.
The substrate is made of a material which is transparent, stable in properties and free from toxic effects on red blood cells. The cover plate is made of a material which is transparent, stable in property and free of toxic effect on red blood cells.
The included angle between the side edges of the first connecting part and the second connecting part is 30-120 degrees, so that the wave channel is smoothly connected with the buffer straight channel.
The longer the path length of the wave channel is within the scope of the microscope CCD recording, the better.
Further, the perfusion liquid propulsion device is adopted to flow the perfusion liquid of the red blood cells into the micro-fluidic chip for detecting the deformability of the red blood cells; the perfusion liquid propelling device comprises an inlet perfusion liquid container, a connecting hose and an outlet perfusion liquid container; perfusion liquid is respectively contained in an inlet perfusion liquid container and an outlet perfusion liquid container, and the liquid levels of the perfusion liquid in the inlet perfusion liquid container and the outlet perfusion liquid container have a height difference h; the bottom ends of the inlet perfusion liquid container and the outlet perfusion liquid container are respectively connected to an inlet through hole and an outlet through hole of the microfluidic chip through connecting hoses, and h is greater than 0.
The invention also aims to provide a preparation method of the microfluidic chip for detecting the deformability of the red blood cells.
The preparation method of the microfluidic chip for detecting the deformability of the red blood cells comprises the following steps:
1) providing a hard template which can be used repeatedly, and forming a male pattern of a wavy micro-flow channel on the template;
2) spreading the stock solution of the substrate on the template to cover the whole male pattern;
3) solidifying the stock solution to form a substrate, thereby forming a negative pattern of a wavy micro-flow channel on the surface of the substrate, wherein the wavy micro-flow channel comprises an inlet, a first buffer straight channel, a first connecting part, a wave channel, a second connecting part, a second buffer straight channel and an outlet which are sequentially connected;
4) punching holes on the surface of the substrate without the intaglio patterns to respectively form an inlet through hole and an outlet through hole which are respectively connected with an inlet and an outlet of the wavy micro-flow channel;
5) the surface of the substrate with the intaglio patterns and the transparent cover plate are sealed together in a bonding mode, so that a closed channel, namely a wave-shaped micro-flow channel, is formed between the surface of the substrate and the cover plate.
The invention also aims to provide a detection method of the microfluidic chip for detecting the deformability of the red blood cells.
The detection method of the microfluidic chip for detecting the deformability of the red blood cells comprises the following steps:
1) inverting the fluorescence microscope, arranging a micro-fluidic chip for detecting the deformability of the red blood cells on the fluorescence microscope, and enabling one side of a cover plate to face downwards to the fluorescence microscope;
2) injecting a perfusion liquid into a micro-fluidic chip for detecting the deformability of the red blood cells by adopting a perfusion liquid propelling device and a pressure gradient method controlled by a height difference, and propelling the flow of the perfusion liquid in the micro-fluidic chip;
3) the light source vertically irradiates the wavy microfluidic channel from one side of the substrate;
4) opening the video recording function of the inverted fluorescence microscope, and recording the video of the red blood cells passing through the wavy microflow channel under the fixed pressure gradient;
5) according to the speed of the red blood cells passing through the wavy microfluidic channel, the deformability of the red blood cells is characterized.
In step 2), in the pressure gradient method, a pressure gradient is defined as a ratio of a pressure difference Δ p between an inlet and an outlet of the perfusion liquid to a length L of the wavy microfluidic channel, where ρ is a density of the perfusion liquid, g is a gravitational acceleration, h is a liquid level difference of the perfusion liquid in an inlet perfusion liquid container and an outlet perfusion liquid container of the perfusion liquid propelling device, and the pressure gradient is Δ p/L and has a unit Pa/μm. When the liquid level is kept flat, namely the height difference is 0, the perfusion liquid in the wavy microfluidic channel is in a static state, and the fixed pressure gradient corresponds to the fixed fluid speed in the microfluidic chip channel. The pressure gradient method is suitable for the condition that the flow rate of perfusion liquid is low: greater than 0 and less than 50000 mu m3The flow rate per hour is 0.18ml, the corresponding page descending height of the perfusion fluid container during the test period is basically negligible, and the perfusion fluid container requires: the syringe capacity used was greater than 20ml so that the liquid surface tension effect could be neglected.
The conventional syringe pump method has a minimum flow unit of about 106μm3S, i.e. only flow rates greater than 10 can be achieved6μm3The pressure gradient method of the invention has the advantages that the height difference h corresponding to the perfusion flow change is a continuous numerical value which is more than or equal to 0, so that the perfusion flow speed regulation precision and the minimum flow speed which can not be achieved by the injection pump can be realized, and the requirement on the CCD frame frequency is also reduced.
In the step 5), the speed of the red blood cells passing through the wavy microfluidic channel is measured under a fixed pressure gradient, and the higher the passing speed, the stronger the deformability, and the weaker the deformation.
The invention has the advantages that:
the invention adopts a wavy microfluidic channel which comprises an inlet, a first buffer straight channel, a first connecting part, a wave channel, a second connecting part, a second buffer straight channel and an outlet which are connected in sequence, and when continuous extrusion stress is provided, the red blood cells are bent and deformed along with the change of waves, so that stress in another form is provided; injecting a perfusion liquid into a micro-fluidic chip for detecting the deformability of the red blood cells by adopting a perfusion liquid propelling device and a pressure gradient method controlled by a height difference, and propelling the flow of the perfusion liquid in the micro-fluidic chip; the invention can achieve the purpose of detecting the deformability of the red blood cells in vitro, can effectively simulate the blood microcirculation environment of human capillaries from the diameter and the shape, and particularly can simulate the extrusion condition of the red blood cells at the spleen in vitro experiment.
Drawings
FIG. 1 is a schematic diagram of one embodiment of a microfluidic chip for detecting deformability of red blood cells according to the present invention;
fig. 2 is a top view of a wavy microfluidic channel of an embodiment of the microfluidic chip for detecting deformability of red blood cells according to the present invention.
Detailed Description
The invention will be further elucidated by means of specific embodiments in the following with reference to the drawing.
As shown in fig. 1, the microfluidic chip for detecting deformability of red blood cells of the present embodiment includes: the device comprises a substrate 1, a wave-shaped micro-flow channel and a cover plate 2; the surface of the substrate 1 with the negative pattern and the transparent cover plate 2 are sealed together in a bonding mode, so that a closed channel, namely a wavy micro-flow channel, is formed between the surface of the substrate and the cover plate; as shown in fig. 2, the wavy microfluidic channel includes an inlet 3, a first buffer straight channel 4, a first connecting portion 41, a wavy channel 5, a second connecting portion, a second buffer straight channel, and an outlet, which are connected in sequence; an inlet through hole and an outlet through hole are respectively formed in the substrate, the inlet through hole is communicated to an inlet of the wavy micro-flow channel, and the outlet through hole is communicated to an outlet of the wavy micro-flow channel. The perfusion liquid propelling device comprises an inlet perfusion liquid container 6, a connecting hose and an outlet perfusion liquid container 7; the bottom ends of the inlet perfusion liquid container and the outlet perfusion liquid container are respectively connected to the inlet through hole and the outlet through hole of the microfluidic chip through connecting hoses.
In the present embodiment, the height of the wavy microfluidic channel is 4 μm; the shape of the plane of the inlet and the outlet is a circle with the diameter of 1 mm; the width of the first and second buffer straight channels is 30 μm for stabilizing the perfusion fluid velocity entering from the inlet; the first and second connecting portions have side angles of 30 °; the shape of the plane of the wave channel comprises 8 periods, each period is two half circles which are connected equally, and the width of each half circle is 3 mu m; the linear distance between the outlet and the inlet is 219 μm long, and the actual path length is 343.8 μm.
The channel with deformability characterized by the "speed of passage of red blood cells" has its own detection limit, whose upper limit of speed detection is expressed as: vmaxRed cell path CCD maximum frame rate. Compared with a straight channel, under the condition that the straight distance between the outlet and the inlet is the same, the wave-shaped channel path is pi/2 times of the original path, and the channel can detect the speedThe limit is also increased by a factor of pi/2.
The preparation method of the microfluidic chip for detecting the deformability of the red blood cells comprises the following steps:
1) providing a hard silicon plate which can be repeatedly used as a template, and forming a male pattern of a wavy micro-flow channel on the template;
2) preparing a stock solution of polydimethylsiloxane PDMS, wherein a prepolymer of PDMS comprises the following basic components in proportion: mixing the curing agents in a ratio of 10:1, fully stirring, vacuumizing, and paving on a template to cover the whole male pattern;
3) drying at 80 ℃ for 40min, and solidifying the stock solution to form a substrate, thereby forming a negative pattern of a wavy microfluidic channel on the surface of the substrate, wherein the wavy microfluidic channel comprises an inlet, a first buffer straight channel, a first connecting part, a wavy channel, a second buffer straight channel of a second connecting part and an outlet which are sequentially connected;
4) punching holes on the surface of the substrate without the intaglio patterns to respectively form an inlet through hole and an outlet through hole which are respectively connected with an inlet and an outlet of the wavy micro-flow channel;
5) the surface of the substrate having the negative pattern and the cover plate of a transparent 0.1mm thick glass were bonded together by oxygen plasma for 5 seconds, thereby forming a closed channel, i.e., a wavy micro-flow channel, between the surface of the substrate and the cover plate.
The detection method of the microfluidic chip for detecting the deformability of the red blood cells comprises the following steps:
1) inverting the fluorescence microscope 9, arranging the microfluidic chip for detecting the deformability of the red blood cells on the fluorescence microscope, and enabling one side of the cover plate to face downwards to the fluorescence microscope;
2) injecting a perfusion liquid into a micro-fluidic chip for detecting red blood cell deformability by adopting a perfusion liquid propelling device and a pressure gradient method controlled by a height difference, wherein the height difference between an inlet perfusion liquid container and an outlet perfusion liquid container 7 is h, and pushing the perfusion liquid in the micro-fluidic chip to flow;
3) the light source 8 vertically irradiates the wavy microfluidic channel from one side of the substrate;
4) opening the video recording function of the inverted fluorescence microscope, and recording the video of the red blood cells passing through the wavy microflow channel under the fixed pressure gradient;
5) according to the speed of the red blood cells passing through the wavy microfluidic channel, the deformability of the red blood cells is characterized.
Finally, it is noted that the disclosed embodiments are intended to aid in further understanding of the invention, but those skilled in the art will appreciate that: various substitutions and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the invention should not be limited to the embodiments disclosed, but the scope of the invention is defined by the appended claims.
Claims (8)
1. A microfluidic chip for detecting deformability of erythrocytes, comprising: the device comprises a substrate, a wave-shaped micro-flow channel and a cover plate; wherein, a positive pattern with a wavy micro-flow channel is formed on the hard template; the stock solution of the substrate is paved on the template to cover the whole male pattern, the base plate is formed after the stock solution is solidified, and the female pattern of the wavy micro-flow channel is formed on the surface of the base plate; the surface of the substrate with the negative pattern and the transparent cover plate are sealed together in a bonding mode, so that a closed channel, namely a wave-shaped micro-flow channel, is formed between the surface of the substrate and the cover plate; the wavy microfluidic channel comprises an inlet, a first buffer straight channel, a first connecting part, a wavy channel, a second connecting part, a second buffer straight channel and an outlet which are connected in sequence, the width of the first buffer straight channel and the width of the second buffer straight channel are larger than the maximum diameter of red blood cells, the height of the first buffer straight channel and the height of the second buffer straight channel are larger than the maximum thickness of the red blood cells, so that the red blood cells are not extruded when passing through, and the first connecting part and the second connecting part are isosceles trapezoids and are larger than the maximum thickness of the red blood cells; the width of the front end of the first connecting part is the same as that of the first buffering direct current channel, the width of the tail end of the first connecting part is the same as that of the wave channel, the width of the front end of the second connecting part is the same as that of the wave channel, and the width of the tail end of the second connecting part is the same as that of the second buffering direct current channel; the width of the wave channel is more than 1 μm and less than the minimum diameter of the red blood cells, and the height of the wave channel is more than 1 μm and less than the minimum diameter of the red blood cells, so that the red blood cells are extruded and deformed when passing through the wave channel, and the wave channel is in a curved shape; an inlet through hole and an outlet through hole are respectively formed in the substrate, the inlet through hole is communicated to an inlet of the wavy micro-flow channel, and the outlet through hole is communicated to an outlet of the wavy micro-flow channel.
2. The microfluidic chip of claim 1, wherein the substrate is made of a material that is transparent to light, stable in properties, and non-toxic to red blood cells.
3. The microfluidic chip according to claim 1, wherein the side edges of the first and second connecting portions are at an angle of 30 to 120 ° to allow smooth connection between the wave channel and the buffer straight channel.
4. The microfluidic chip according to claim 1, further comprising a perfusion propulsion device comprising an inlet perfusion container, a connection hose, and an outlet perfusion container; perfusate is respectively contained in the inlet perfusate container and the outlet perfusate container, the inlet perfusate container and the outlet perfusate container are positioned on the microfluidic chip, and the liquid level of the perfusate in the inlet perfusate container and the liquid level of the perfusate in the outlet perfusate container have a height difference h; the bottom ends of the inlet perfusion liquid container and the outlet perfusion liquid container are respectively connected to an inlet through hole and an outlet through hole of the microfluidic chip through connecting hoses, and h is greater than 0.
5. A preparation method of a microfluidic chip for detecting erythrocyte deformability is characterized by comprising the following steps:
1) providing a hard template which can be used repeatedly, and forming a male pattern of a wavy micro-flow channel on the template;
2) spreading the stock solution of the substrate on the template to cover the whole male pattern;
3) solidifying the stock solution to form a substrate, thereby forming a negative pattern of a wavy micro-flow channel on the surface of the substrate, wherein the wavy micro-flow channel comprises an inlet, a first buffer straight channel, a first connecting part, a wave channel, a second connecting part, a second buffer straight channel and an outlet which are sequentially connected;
4) punching holes on the surface of the substrate without the intaglio patterns to respectively form an inlet through hole and an outlet through hole which are respectively connected with an inlet and an outlet of the wavy micro-flow channel;
5) the surface of the substrate with the negative pattern and the transparent cover plate are bonded together through oxygen plasma, so that a closed channel, namely a wave-shaped micro-flow channel, is formed between the surface of the substrate and the cover plate.
6. A detection method of a microfluidic chip for detecting erythrocyte deformability is characterized by comprising the following steps:
1) inverting the fluorescence microscope, arranging a micro-fluidic chip for detecting the deformability of the red blood cells on the fluorescence microscope, and enabling one side of a cover plate to face downwards to the fluorescence microscope;
2) injecting a perfusion liquid into a micro-fluidic chip for detecting the deformability of the red blood cells by adopting a perfusion liquid propelling device and a pressure gradient method controlled by a height difference, and propelling the flow of the perfusion liquid in the micro-fluidic chip;
3) the light source vertically irradiates the wavy microfluidic channel from one side of the substrate;
4) opening the video recording function of the inverted fluorescence microscope, and recording the video of the red blood cells passing through the wavy microflow channel under the fixed pressure gradient;
5) according to the speed of the red blood cells passing through the wavy microfluidic channel, the deformability of the red blood cells is characterized.
7. The detection method according to claim 6, wherein in the step 2), in the pressure gradient method, the pressure gradient is defined as a ratio of a pressure difference Δ p ═ ρ gh between an inlet and an outlet of the perfusion fluid to a length L of the wavy microfluidic channel, wherein ρ is a density of the perfusion fluid, g is a gravitational acceleration, h is a height difference of liquid levels of the perfusion fluid in an inlet perfusion fluid container and an outlet perfusion fluid container of the perfusion fluid propulsion device, and the pressure gradient is Δ p/L in Pa/μm.
8. The detection method as claimed in claim 6, wherein in step 5), the speed of the red blood cells passing through the wavy microfluidic channel is measured under a fixed pressure gradient, and the higher the passing speed, the stronger the deformability is, and the weaker the deformability is.
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