阅读说明：本技术 一种用于活细胞力学性能高通量精密检测的装置及方法 (Device and method for high-throughput precision detection of mechanical properties of living cells ) 是由 陈华英 陈震林 张露丹 朱永刚 于 2021-08-23 设计创作，主要内容包括：本发明公开了一种用于活细胞力学性能高通量精密检测的微流控装置,用于检测活细胞力学性能的微流体芯片置于光学显微镜上方,微流体芯片内设置并列的微通道用于对细胞进行挤压,并且设置分流通道保证挤压压力基本不变；一种用于活细胞力学性能高通量精密检测的方法,包括以下步骤：S1：细胞弹性模量的测量方法；S2：微通道压差控制方法；该装置及方法不但可以快速检测大量活细胞的弹性模量,而且操作简单,高度自动化。(The invention discloses a microfluidic device for high-flux precision detection of mechanical properties of living cells, wherein a microfluidic chip for detecting the mechanical properties of the living cells is arranged above an optical microscope, parallel microchannels are arranged in the microfluidic chip and used for extruding the cells, and a shunting channel is arranged to ensure that the extrusion pressure is basically unchanged; a method for high-throughput precision detection of mechanical properties of living cells comprises the following steps: s1: a method for measuring the cell elastic modulus; s2: a microchannel differential pressure control method; the device and the method not only can rapidly detect the elastic modulus of a large number of living cells, but also have simple operation and high automation.)

1. A device for high-flux precision detection of mechanical properties of living cells is characterized by comprising a microfluid chip and an optical microscope, wherein the microfluid chip is arranged above the optical microscope, and parallel microchannels are arranged in the microfluid chip and used for extruding cells;

the microfluid chip includes entry, export, is located middle part extrusion district's microchannel and reposition of redundant personnel passageway side by side, the opposite side of entry sets up the fillter section, the opposite side of fillter section sets up the reposition of redundant personnel passageway, the both sides in extrusion district set up the reposition of redundant personnel passageway respectively, the inside microchannel that sets up in extrusion district, the entry setting of reposition of redundant personnel passageway is the microchannel side by side, the opposite side of reposition of redundant personnel passageway sets up the export.

2. The device for high-throughput precision detection of mechanical properties of living cells according to claim 1, wherein parallel microchannels are provided at the entrance of the shunting channel for preventing cells from entering the shunting channel, the width of the parallel microchannels in the extrusion region is smaller than the diameter of the cells for forcing the cells to deform, and the pressure drop across the microchannels in the extrusion region is kept constant.

3. A method for high-throughput precision detection of mechanical properties of living cells is characterized by comprising the following steps:

s1: a microchannel differential pressure control method;

s2: a high-throughput and high-precision measurement method of cell elastic modulus.

4. The method for high-throughput precision detection of mechanical properties of living cells according to claim 3, wherein the S1 comprises the following sub-steps:

s11: the chip is connected with an external differential pressure sensor and used for determining an accurate pressure drop value of the extrusion area;

s12: injecting a cell-free liquid into the chip at a flow rate using a precision syringe pump or pressure pump, the sensor detecting the pressure drop in the constricted passage of the expression region and not measuring it in a subsequent step, the pressure drop remaining substantially constant as the cells pass through the constricted passage;

s13: introducing suspension containing cells to be detected into the microfluidic chip at the same flow rate;

s14: most cells in the middle of the channel flow to the micro-channel of the extrusion region along the flow line, and individual cells close to the wall surface flow to the inlet of the shunt channel along the channel wall surface;

s15: because the micro-channels at the inlets of the extrusion area and the flow distribution channel are very narrow and the width of the micro-channels is far smaller than that of the extrusion area and the flow distribution channel, the pressure drop of the chip is mainly concentrated at the micro-channels;

s16: when the cells reach the inlet of the shunting channel, the width of the microchannel at the inlet is far smaller than the diameter of the cells, so that the cells are not easy to deform and cannot enter the shunting channel, and flow to a downstream extrusion area along the wall surface under the action of a fluid;

s17: under the partial pressure action of the parallel micro-channels of the extrusion area and the shunt channel, the whole pressure change of the channels is very small and can be ignored in the process of extruding cells by the micro-channels of the extrusion area, so that the high precision of elastic modulus detection is ensured.

5. The method for high-throughput precision detection of mechanical properties of living cells according to claim 3, wherein the S2 comprises the following sub-steps:

s21: the suspension containing the living cells to be detected is injected into the main channel from the inlet of the chip at a certain flow rate by an external precise injection pump or a pressure pump;

s22: the cells continue to flow to a downstream extrusion area under the action of the fluid, and when the cells flow to the extrusion area micro-channel, the cells are extruded and deformed to enter the micro-channel because the width of the contraction channel is smaller than the diameter of the cells;

s23: the cells flow downstream after passing through the microchannel and flow out of the chip from the outlet;

s24: 40 microchannels are distributed in the channel in parallel, so that the cells can flow through the channel in a high flux, and simultaneously all the microchannels can be observed by an optical microscope;

s25: the cell deformation condition in the micro-channel is recorded by a microscopic imaging system and quantified cell deformation data is obtained;

s26: thanks to the shunting arrangement of the plurality of parallel micro-channels and the side shunting channels, in the cell extrusion process, the number of the blocked micro-channels is controlled by controlling the number of the simultaneously extruded cells so as to obtain high-precision and stable pressure drop, and ensure the high precision and high flux of the cell mechanical property detection.

Technical Field

The invention relates to the field of cell biology, in particular to a device and a method for high-throughput precision detection of mechanical properties of living cells.

Background

The elastic modulus of a cell influences various aspects of cell proliferation, differentiation, migration, apoptosis and the like. The abnormal elastic modulus of the cells may cause the change of the cell functions and even cause the disease. For example, when humans are infected with Plasmodium falciparum, the elastic modulus of erythrocytes in humans increases. The difficulty of the hardened red blood cells in passing through the narrow capillaries results in poor blood flow and may eventually lead to coma and even death of the person. In addition, in stem cell therapy, safety and effectiveness are limited by various factors such as pulmonary embolism due to cell clogging in capillaries and insufficient cell activity and purity before implantation, and these problems are closely related to the elastic modulus of cells. Therefore, studying the elastic modulus of cells can be used to quantitatively reflect the health status of cells, and is expected to be useful for rapid diagnosis and treatment of diseases.

Measurement studies of the modulus of elasticity of cells have been carried out for half a century, and techniques such as optics, magnetism, and hydrodynamics have been applied to cell mechanics studies. The most widely used conventional measurement technique is a Micropipette (Micropipette agitation). This technique utilizes aspiration of spherical cells through a glass capillary and obtains the elastic modulus of the cells based on cell deformation data. Atomic Force Microscopy (Atomic Force Microscopy) is currently the most accurate technique for measuring the elastic modulus of single cells. The technique uses a precision cantilever free end to press against the cell, causing a local deformation of the cell surface and obtaining the elastic modulus of the cell from the deformation and the required force. However, the atomic force microscope equipment is high in price, complex to operate and very low in measurement efficiency. Besides, the conventional cell elastic modulus measurement technologies include Magnetic twist Cytometry (Magnetic twist Cytometry), Optical Tweezers (Optical Tweezers) and Shear Flow technology (Shear Flow) [8], but these technologies also have corresponding problems, such as the problem of calibrating the magnitude of the extrusion force in the Optical Tweezers technology.

The above conventional measurement techniques all have some disadvantages, such as low throughput, expensive equipment or cumbersome operation. Because of the large individual differences in cells, it is desirable to try to measure a large number of cells to obtain statistically reliable data. Microfluidic technology has the potential for high throughput measurement of the elastic modulus of living cells because it can process cells at high throughput. Related studies have emerged to measure the elastic modulus of cells by microfluidic techniques. Flow induced deformation (Flow induced deformation) techniques utilize shear Flow in microchannels to induce cell deformation and estimate its elastic modulus. Gossett et al used this technique to calculate cell deformability and achieved a flux of approximately 2000 cells per second. The real-time cell deformation technique developed by Otto et al also used this technique to quantitatively calculate the elastic modulus of the cells and achieved a throughput of approximately 100 cells per second. Compression (Compression) techniques use deformable membranes in microfluidic chips to compress and deform cells to obtain elastic modulus data. Hohne et al measured the mechanical properties of cells with Young's modulus in the range of 102-105Pa using this technique. In addition, the micropipette (micropipette) technique, similar to the micropipette in the conventional measurement method, allows the cell to be pressed and deformed in the micro channel to obtain elastic modulus data. The technique records the deformation process of the cells in a micro-fluidic device by combining with a micro-imaging technology, thereby obtaining the elastic modulus of the cells. Guo et al developed micron-scale conical constriction channels to deform cells through and thereby estimate the deformability of cells. Kim et al passed cells through a plurality of parallel funnel-shaped constricting microchannels in a microfluidic chip and deformed to obtain the elastic modulus of the cells.

In summary, the background art has significant disadvantages compared to the present invention. Briefly summarized as follows:

1) although the elastic modulus measurement of the atomic force microscope is high in precision, the flux is low, and a large amount of data with statistical significance is difficult to obtain. Real-time deformable cytometry (Real-time deformable cytometry) based on flow-induced deformation has the disadvantages of limited induction force, inability to deform harder cells, and the need for a high-speed camera to capture cell deformation data, although throughput is high.

2) Although the existing microfluidic device can process a plurality of single cells in parallel, the pressure difference at two ends of a cell deformation channel is not precisely measured and controlled, and the situation that the pressure difference is severely changed due to the blockage of all contraction channels can occur, so that the calculated value of the elastic modulus of the cells has huge errors.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a device and a method for high-throughput precision detection of mechanical properties of living cells, which not only can quickly detect the elastic modulus of a large number of living cells, but also has simple operation and high precision.

A device for high-flux precision detection of mechanical properties of living cells comprises a microfluid chip and an optical microscope, wherein the microfluid chip is arranged above the optical microscope, and parallel microchannels are arranged in the microfluid chip and used for extruding cells;

the microfluid chip includes entry, export, is located the microchannel and the reposition of redundant personnel passageway side by side of middle part extrusion district, the opposite side of entry sets up the fillter section, and the opposite side of fillter section sets up the reposition of redundant personnel passageway, and the both sides in extrusion district set up the reposition of redundant personnel passageway respectively, and the inside microchannel that sets up in extrusion district, the opposite side of reposition of redundant personnel passageway sets up the export.

Preferably, parallel microchannels with the width of 4 microns are arranged at the inlet of the shunting channel to prevent the cells from entering the shunting channel, the width of the parallel microchannels of the extrusion area is smaller than the diameter of the cells to force the cells to deform, and the pressure drop between the upstream and the downstream of the microchannels in the extrusion area is kept at delta P.

Preferably, the method for high-throughput precision detection of mechanical properties of living cells comprises the following steps:

s1: a microchannel differential pressure control method;

s2: a high-throughput and high-precision measurement method of cell elastic modulus.

Preferably, S1 includes the following sub-steps:

s11: the chip is connected with an external differential pressure sensor and used for determining an accurate pressure drop value of the extrusion area;

s12: injecting a cell-free liquid into the chip at a flow rate using a precision syringe pump or pressure pump, the sensor detecting a pressure drop in the constricted passage in the extrusion region, the pressure drop remaining substantially constant as the cells pass through the constricted passage;

s13: introducing suspension containing cells to be detected into the microfluidic chip at the same flow rate;

s14: most cells in the middle of the channel flow to the micro-channel of the extrusion region along the flow line, and individual cells close to the wall surface flow to the inlet of the shunt channel along the channel wall surface;

s15: because the micro-channels at the inlets of the extrusion area and the flow distribution channel are very narrow, and the width of the main channel is more than 100 times of that of the micro-channel, the pressure drop of the channel is mainly concentrated at the micro-channel;

s16: when the cells reach the inlet of the shunting channel, the width of the microchannel at the inlet is far smaller than the diameter of the cells, so that the cells are not easy to deform and cannot enter the shunting channel, and flow to a downstream extrusion area along the wall surface under the action of a fluid;

s17: under the partial pressure action of the micro-channel, the whole pressure change of the channel is tiny and can be ignored in the process of extruding the cells by the micro-channel in the extrusion area, so that the high precision of elastic modulus detection is ensured;

preferably, S2 includes the following sub-steps: :

s21: the suspension containing the living cells to be detected is injected into the main channel from the inlet of the chip at a certain flow rate by an external precise injection pump or a pressure pump;

s22: the cells continue to flow to a downstream extrusion area under the action of the fluid, and when the cells flow to the extrusion area micro-channel, the cells are extruded and deformed to enter the micro-channel because the width of the contraction channel is smaller than the diameter of the cells;

s23: the cells flow downstream after passing through the microchannel and flow out of the chip from the outlet;

s24: 40 microchannels are distributed in the channel in parallel, so that the cells can flow through the channel in a high flux, and simultaneously all the microchannels can be observed by an optical microscope;

s25: the cell deformation condition in the micro-channel is recorded by a microscopic imaging system and quantified cell deformation data is obtained;

s26: owing to the shunting arrangement of the plurality of parallel micro-channels and the side shunting channels, high-precision and stable pressure drop can be obtained in the cell extrusion process, and high precision and high flux of cell mechanical property detection are ensured.

The device and the method for high-throughput precision detection of the mechanical property of the living cells have the following beneficial effects:

1. by arranging the parallel extrusion channels and realizing constant pressure difference in the detection process, the cell elastic modulus detection flux is improved, a large amount of cell elastic modulus data can be rapidly provided, and the commercial application of the cell elastic modulus chip becomes possible.

2. Based on the arrangement of the flow channel, the stability of the pressure difference in the measuring process is strictly ensured, so that the precision measurement of the cell elastic modulus is realized.

Drawings

FIG. 1 is a schematic diagram of a cell elastic modulus detection device.

FIG. 2 is a schematic view of a microfluidic chip channel and a partially enlarged view of a constriction channel.

FIG. 3 cell extrusion and microchannel magnification

Fig. 4 is a flow chart of elastic modulus measurement.

FIG. 5(a) channel streamline distribution diagram;

FIG. 5(b) schematic view of the inlet of the shunt channel blocking cells.

FIG. 6(a) a deformation map of cells in the squeezed area;

FIG. 6(b) is a diagram of cell deformation in a single microchannel in the extrusion zone;

FIG. 6(c) cell elongation as a function of time and fitting results according to cell model (solid line).

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

As shown in fig. 1-3, the device consists essentially of a microfluidic chip and a microscopic imaging system, and uses parallel microchannels in the microfluidic chip to compress cells. The internal flow channel of the microfluidic chip is shown in fig. 3 and comprises an inlet, an outlet, parallel microchannels located in the middle extrusion region and flow splitting channels located on two sides. The width of the parallel micro-channel in the extrusion area is smaller than the diameter of the cell, so that the cell can be forced to deform, and the micro-channel is arranged at the inlet of the shunt channel and used for preventing the cell close to the wall surface from entering the shunt channel. Under the shunting action of the parallel channels and the shunting channels, the pressure drop of the extrusion area is basically changed negligibly in the process of extruding the cells.

The elastic modulus measurement chip uses a flow chart as shown in fig. 4.

Before the elastic modulus measurement, a liquid without cells was injected into the chip at a certain flow rate using a precision syringe pump, and the precise pressure drop values of the squeezing zone at different flow rates were measured using an external differential pressure sensor.

Subsequently, a suspension containing the cells to be tested is passed over the microfluidic chip using a precision syringe pump or a pressure pump, and the cell deformation at the microchannel is recorded using a microscopic imaging system.

As shown in FIG. 5a, when a suspension containing the living cells to be measured flows into the main channel from the inlet of the chip by the external precision syringe pump at a certain flow rate, since the cells in the chip flow along the streamline, most of the cells in the middle of the channel will flow along the streamline to the microchannel of the extrusion region, and the individual cells will flow along the channel wall surface to the inlet of the shunt channel.

When the cell reaches the entrance of the shunting channel, the cell is not easily deformed and cannot enter the shunting channel because the width of the 4 micron microchannel at the entrance is much smaller than the diameter of the cell, as shown in fig. 5 b.

The cells then continue to flow downstream to the extrusion region under the influence of the fluid, and as the cells flow to the extrusion region microchannel, the cells will deform and enter the microchannel as the width of the constricted channel is less than the diameter of the cells.

The cells flow downstream after passing through the microchannel and exit the chip through the outlet.

40 microchannels are distributed in the channel in parallel, so that the cells can flow through the channel at high flux, and all the microchannels can be observed simultaneously through a microscopic imaging system.

The cell deformation in the microchannel is recorded by an optical microscope and quantified cell deformation data is obtained.

Thanks to the flow distribution of the plurality of parallel microchannels and the side flow distribution channels, when the microchannels of the extrusion region are blocked due to cell extrusion, the blocking has a very small influence on the flow resistance of the whole channel, so that the pressure drop of the microchannels of the extrusion region is kept substantially constant during the cell extrusion process.

And calculating the elastic modulus of the cells based on the differential pressure of the micro-channel and the cell deformation amount obtained by the measurement. The radius of each micro-channel in the extrusion area is expressed by the radius of a circular tube with equal cross section areaWhere h and w represent the channel height and width, respectively. And obtaining the elongation of the cell to be detected in the contraction channel by a microscopic imaging system. Then, combining the differential pressure of the contraction channel, and utilizing the cell power law model deformation formulaCalculating the modulus of elasticity, whereinTo shrink the channel coefficient, R_PIs the channel radius, Δ P is the channel pressure difference, L is the cell elongation, α is the power law index, A_JIs the shear modulus. When the deformation time t is equal to t₀1s, elastic modulus of cell A_GCan be according to the formulaAnd (6) calculating. Finally, the elastic modulus of each cell passing through the constriction channel is measured.

As shown in FIG. 6a, as the cells flow into the extrusion region, several cells are continuously deformed and elongated on the parallel micro-channels. Wherein a deformation image of each cell is extracted and the change of cell elongation L with time is measured, as shown in FIG. 6b and6 c. Finally, according to the deformation formula of the cell power law model Cell deformation data was used to fit and obtain the elastic modulus of the cells (as shown by the solid line in fig. 6 c).