CN110523447B

CN110523447B - Microfluidic chip for multi-angle mechanical measurement of cells and manufacturing method thereof

Info

Publication number: CN110523447B
Application number: CN201910811048.3A
Authority: CN
Inventors: 杨浩; 朱博韬; 程亮; 李相鹏; 李婉婷; 孙研珺
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2019-08-29
Filing date: 2019-08-29
Publication date: 2021-05-11
Anticipated expiration: 2039-08-29
Also published as: CN110523447A

Abstract

The invention discloses a microfluidic chip for multi-angle mechanical measurement of cells and a manufacturing method thereof, wherein the microfluidic chip is formed by bonding and packaging a PDMS cover plate provided with a microchannel and an ITO glass substrate etched with an electrode, can be used for multi-angle mechanical measurement of cells under a non-contact condition, and has the advantages of low cost, quick measurement, small damage to the cells and the like; the manufacturing method is simple, the production efficiency is high, the batch production can be realized, and the method has wide commercial application prospect.

Description

Microfluidic chip for multi-angle mechanical measurement of cells and manufacturing method thereof

Technical Field

The invention relates to a microfluidic chip and manufacturing thereof, in particular to a microfluidic chip for multi-angle mechanical measurement of cells and a manufacturing method thereof.

Background

The Microfluidics (Microfluidics) technology is one of the important branches of the MEMS technology, and integrates basic operation units of sample preparation, reaction, separation, detection, etc. in the biological, chemical, and medical analysis processes into a micron-scale chip, thereby automatically completing the whole analysis process. Due to its great potential in the fields of biology, chemistry, medicine and the like, it has been developed into a new research field crossing the subjects of biology, chemistry, medicine, fluid, electronics, materials, machinery and the like, is one of the advanced technologies of multidisciplinary cross science and technology which are developed rapidly at present, and has important application in the subjects of life science, clinical medicine, chemical engineering, pharmacy, food sanitation, environmental detection and monitoring, information science, signal detection and the like.

Micro-fluidic technology generally uses micro-analysis devices as carriers for technical implementation, and micro-fluidic chips are the most rapidly developed among various types of micro-analysis devices. The micro-fluidic chip is a micro total analysis system which utilizes the MEMS technology to process various microstructures on a silicon, quartz, glass or high molecular polymer substrate, such as functional units of a micro pipeline, a micro reaction pool, a microelectrode and the like, and then uses the micro pipeline to communicate components with fluid conveying, controlling, detecting and monitoring functions, such as a micro pump, a micro valve, a micro liquid storage device, a micro detection element and the like, so as to furthest integrate the processes of diluting, adding a reagent, sampling, reacting, separating, dispersing, detecting, monitoring and the like on the chip. The area of the microfluidic chip is usually several to tens of square centimeters, and the size of the microchannel is generally in the micrometer or near millimeter level. The microfluidic chip has the advantages of low cost, batch manufacturing, simple operation, good repeatability, high reliability and the like.

At present, the widely accepted biological chips (micro-arrays), such as gene chips, protein chips and the like, are only point array type hybridization chips with zero micro-flow, have very limited functions, belong to the special types of micro-fluidic chips (micro-chips), have wider types, functions and applications, can develop analysis systems such as biological computers, gene and protein sequencing, mass spectrometry, chromatography and the like, and become an extremely important technical basis of system biology, particularly system genetics. Although there are many kinds of microfluidic chips and applications thereof, there is no microfluidic chip for performing multi-angle mechanical measurement on cells.

Disclosure of Invention

In view of this, the present invention provides a microfluidic chip for multi-angle mechanical measurement of cells, so as to make up for the disadvantages of the existing types and applications of microfluidic chips.

The microfluidic chip for multi-angle mechanical measurement of cells is formed by packaging a substrate and a cover plate, wherein the cover plate is Polydimethylsiloxane (PDMS) provided with a microchannel, AB sections and AC sections of the microchannel are symmetrically arranged relative to AD sections positioned on a central line, AE sections are linear extension sections of the AD sections, the AB sections and the AC sections have the same width, the AD sections and the AE sections have the same width and are wider than the AB sections and the AC sections, a first liquid inlet, a second liquid inlet, a third liquid inlet and a liquid outlet are respectively and correspondingly arranged at a B end, a C end, a D end and an E end of the microchannel, and BAC is 30-60 degrees; the substrate is ITO (indium tin oxide) glass with the size slightly larger than that of the cover plate, electrodes are etched on the indium tin oxide layer of the substrate, are positioned in the AE section of the micro-channel and are tightly bonded with the AE section of the micro-channel, and comprise an upper electrode and two lower electrodes which are mutually spaced.

In the technical scheme, the width ratio of the AB section to the AC section to the AD section to the AE section of the micro-channel is 1: 2.0-3.0.

Furthermore, the widths of the AB section and the AC section of the micro-channel are 200 micrometers, and the widths of the AD section and the AE section are 450 micrometers.

In the technical scheme, the angle BAC is 60 degrees.

In the technical scheme, the distance between the upper electrode and the lower electrode is 30-60 micrometers, and the distance between the two lower electrodes is 15-30 micrometers.

The invention also provides a manufacturing method of the microfluidic chip for the multi-angle mechanical measurement of the cells, which comprises the following steps:

(1) etching an upper electrode and two lower electrodes which are mutually spaced on an indium tin oxide layer of the ITO glass to be used as substrates;

(2) photoetching to obtain a positive membrane, reversing the mold to obtain a PDMS microchannel, wherein an AB section and an AC section of the microchannel are symmetrically arranged relative to an AD section positioned on a central line, an AE section is a linear extension section of the AD section, the AB section and the AC section have the same width, the AD section and the AE section have the same width and are wider than the AB section and the AC section, a B end, a C end, a D end and an E end of the microchannel are respectively and correspondingly provided with a first liquid inlet, a second liquid inlet, a third liquid inlet and a liquid outlet, and angle BAC is 30-60 degrees (the angle BAC represents an included angle formed between the AB section and the AC section);

the specific manufacturing process of the PDMS micro-channel is as follows:

coating photoresist on a silicon wafer;

baking the silicon wafer coated with the photoresist;

carrying out photoetching process on the baked silicon wafer through a preset mask plate to obtain a micro-channel mold;

enclosing the periphery of the silicon wafer to form a box-shaped cavity;

pouring resin solution into the box-shaped cavity;

and after the resin solution is solidified, separating the resin solution from the silicon wafer to obtain the micro-flow channel layer containing the micro-flow channel.

3) And aligning the ITO glass substrate and the PDMS cover plate through an alignment platform to enable the electrode to be positioned in the AE section of the micro-channel, and tightly bonding to obtain the micro-fluidic chip.

In the technical scheme, the angle BAC is 60 degrees.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a micro-fluidic chip capable of performing multi-angle mechanical measurement on cells for the first time, which can realize the capture of the cells by an electrode by controlling the width ratio and the angle of a micro-channel, stretch the cells in different directions, perform multi-angle mechanical measurement on the cells under a non-contact condition, can be repeatedly used after being cleaned, and has the advantages of low cost, rapid measurement, small damage to the cells and the like. The manufacturing method of the micro-fluidic chip is simple, has high production efficiency, can realize batch production, and has wide commercial application prospect.

Drawings

FIG. 1 is a schematic structural view of unprocessed ITO glass;

FIG. 2 is a schematic structural diagram of an ITO glass substrate after electrode etching;

FIG. 3 is an enlarged view of a portion of an electrode of the ITO glass substrate of FIG. 2;

FIG. 4 is a schematic diagram of a PDMS structure with fabricated microchannels;

FIG. 5 is a schematic diagram of a three-dimensional structure of a bonded and packaged microfluidic chip;

FIG. 6 is a top view of a bond encapsulated microfluidic chip;

FIG. 7 is a schematic diagram of the internal structure of a bonded encapsulated microfluidic chip;

wherein, 1 is an ITO glass substrate, 2 is an electrode, 21 is an upper electrode, 22/23 is a lower electrode, 3 is a cover plate, 4 is a micro-channel, 51 is a first liquid inlet, 52 is a second liquid inlet, 53 is a third liquid inlet, and 6 is a liquid outlet.

Detailed Description

The technical solutions in the embodiments of the present invention will be described in detail below with reference to the accompanying drawings in the embodiments of the present invention.

Example 1

As shown in fig. 1-7, the ITO glass is a conductive glass manufactured by coating a layer of indium tin oxide (commonly referred to as ITO) film on a soda-lime-based or silicon-boron-based substrate glass by various methods such as sputtering and evaporation. By using the processes of photoetching and the like and wet etching technology, the ITO glass is made into the electrode 2 of the invention to be used as the substrate 1, namely, a layer of indium tin oxide on the ITO glass substrate 1 is made into the shape required by the invention by using the soft photoetching process and the etching technology (the traditional mature technology), the electrode 2 comprises an upper electrode 21 and two

lower electrodes

22 and 23 which are mutually spaced, the distance between the upper electrode and the lower electrode is 30 micrometers, and the distance between the two

lower electrodes

22 and 23 is 15 micrometers. Polydimethylsiloxane (PDMS) is one of organic silicon, and is a polymer material widely applied to the fields of microfluidics and the like due to the characteristics of low cost, simple use, good adhesion with a silicon wafer, good chemical inertness and the like. The microchannel 4 is manufactured by PDMS (polydimethylsiloxane) (PDMS) (through a soft lithography process) and is used as a cover plate 3, wherein the AB section and the AC section of the microchannel are symmetrically arranged relative to the AD section positioned on a central line, the AE section is a linear extension section of the AD section, the AB section and the AC section have the same width, the AD section and the AE section have the same width and are wider than the AB section and the AC section, the B end, the C end, the D end and the E end of the microchannel are respectively and correspondingly provided with a first liquid inlet 51, a second liquid inlet 52, a third liquid inlet 53 and a liquid outlet 6, the angle BAC is 30 degrees, the widths of the AB section and the AC section of the microchannel are 200um, and the widths of the AD section and the AE section are 400 um. The ITO glass substrate 1 is placed at the lower part, the PDMS cover plate 3 is placed at the upper part, the alignment platform is used for alignment, the close bonding is carried out to prevent liquid leakage, and the electrode 2 is just positioned in the AE section of the micro-channel 4.

Example 2

The indium tin oxide layer on the ITO glass substrate 1 is made into the required shape of the invention by a soft photoetching process and an etching technology, the electrode 2 comprises an upper electrode 21 and two

lower electrodes

22 and 23 which are mutually spaced, the distance between the upper electrode and the lower electrode is 60 micrometers, and the distance between the two

lower electrodes

22 and 23 is 30 micrometers. The shape of the microchannel 4 is made from PDMS through a soft lithography process and is used as a cover plate 3, wherein the AB section and the AC section of the microchannel are symmetrically arranged relative to the AD section positioned on the central line, the AE section is a linear extension section of the AD section, the AB section and the AC section have the same width, the AD section and the AE section have the same width and are wider than the AB section and the AC section, the B end, the C end, the D end and the E end of the microchannel are respectively and correspondingly provided with a first liquid inlet 51, a second liquid inlet 52, a third liquid inlet 53 and a liquid outlet 6, the angle BAC is 45 degrees, the widths of the AB section and the AC section of the microchannel are 200um, and the widths of the AD section and the AE section are 600 um. The ITO glass substrate 1 is placed at the lower part, the PDMS cover plate 3 is placed at the upper part, the alignment platform is used for alignment, the close bonding is carried out to prevent liquid leakage, and the electrode 2 is just positioned in the AE section of the micro-channel 4.

Example 3

lower electrodes

22 and 23 which are mutually spaced, the distance between the upper electrode and the lower electrode is 45 micrometers, and the distance between the two

lower electrodes

22 and 23 is 20 micrometers. The shape of a microchannel 4 manufactured by PDMS is used as a cover plate 3 through a soft lithography process, wherein a microchannel AB section and an AC section are symmetrically arranged relative to an AD section positioned on a central line, the AE section is a linear extension section of the AD section, the AB section and the AC section have the same width, the AD section and the AE section have the same width and are wider than the AB section and the AC section, a first liquid inlet 51, a second liquid inlet 52, a third liquid inlet 53 and a liquid outlet 6 are respectively and correspondingly arranged at the B end, the C end, the D end and the E end of the microchannel, the angle BAC is 60 degrees, the widths of the microchannel AB section and the AC section are 200um, and the widths of the AD section and the AE section are 450 um. The ITO glass substrate 1 is placed at the lower part, the PDMS cover plate 3 is placed at the upper part, the alignment platform is used for alignment, the close bonding is carried out to prevent liquid leakage, and the electrode 2 is just positioned in the AE section of the micro-channel 4.

Example 4

As shown in fig. 7, the microfluidic chip manufactured in example 3 was used to capture cells and perform multi-angle mechanical measurements, and the included angles between the microchannel AB and the microchannel AC and the microchannel AD were all 30 degrees. The method comprises the following steps of opening a micro-flow pump to flow DEP buffer solution containing cells into a micro-channel AD at a flow rate of 15ul/min (DEP is dielectrophoresis, also called dielectrophoresis, and is a phenomenon that an object with a low dielectric constant is stressed in a non-uniform electric field, the magnitude of the dielectric force is independent of whether the object is electrified or not, and is dependent on the size, the electrical property of a surrounding medium, the field intensity of an external electric field, the field intensity change rate and the frequency of the external electric field. Thus, due to the laminar flow (laminar flow is a flow condition of a fluid which flows in layers, the fluid exhibits laminar flow at low flow velocities in the tube, the particles of which move smoothly in a straight line parallel to the tube axis, the flow velocity of the fluid is greatest at the center of the tube and smallest near the wall, the ratio of the average flow velocity to the maximum flow velocity of the fluid in the tube is equal to 0.5), most of the cells are collected in the middle of the microchannel DE, facilitating the capture by the electrodes.

Secondly, the sine wave of 2Mhz and 2Vpp is passed between the upper electrode 21 and the lower electrode 22, and the cells flow from right to left, so the cells contact the upper electrode 21 first when flowing, and are attracted and captured by the upper electrode 21, and the electrode conduction method can improve the probability of the cells being captured on the upper electrode 21. When a cell is captured on the upper electrode 21, the micro flow pump is turned off, any solution injection is stopped, and the energization between the upper electrode 21 and the lower electrode 22 is stopped.

Then, the mechanical characteristics of the cells can be measured in multiple angles by increasing the voltage between the upper electrode 21 and the lower electrode 22 from 2Vpp to 8Vpp (the cells lose their activity due to too high voltage), then passing the sine wave from 2Vpp to 8Vpp between the upper electrode 21 and the lower electrode 23, and finally, connecting the lower electrode 22 and the lower electrode 23 to the signal generator, connecting the upper electrode 21 to the other end, and increasing the voltage from 2Vpp to 8Vpp again, so that there are 3 sets of data, each of which is the cells stretched in different directions.

After the experiment is finished, absolute ethyl alcohol and air can be sequentially introduced to clean the chip, so that the chip can be conveniently used later.

Specifically, the multi-angle mechanical property measurement analysis is carried out on the mouse red blood cells.

First, DEP buffer containing erythrocytes was introduced at a flow rate of 10ul/min, and a sine wave with a peak value of 4Vpp and a frequency of 1.5MHz was applied to the upper electrode 21 and the lower electrode 22, and when one erythrocyte was captured on the upper electrode 21, the microflow pump was turned off, and the injection of DEP buffer was stopped.

At this time, the voltage was increased on the upper electrode 21 and the lower electrode 22 from 4Vpp to 8Vpp, and the deformation of the red blood cells was recorded. Then the voltage is increased on the upper electrode 21 and the lower electrode 23 from 3Vpp to 8Vpp and the deformation of the red blood cells is recorded. Finally, the upper electrode 21 was connected to the positive electrode and the

lower electrodes

22 and 23 were connected to the negative electrode, while the voltage was increased from 4 to 8Vpp, and the deformation of the red blood cells was recorded.

Data: upper electrode 21 and lower electrode 22, the cell shape change increased from 0.12 to 0.26.

Upper electrode 21 and lower electrode 23, the cell shape change increased from 0.13 to 0.25.

The cell shape change increased from 0.18 to 0.35 for the upper electrode 21 and the

lower electrodes

22 and 23.

And (3) analysis: the upper electrode 21 and the lower electrode 22, and the upper electrode 21 and the lower electrode 23 are energized, respectively, and the amount of cell deformation is almost the same. When energized alone, the cells are subjected to the same DEP force, but in different directions. The upper electrode 21 and the

lower electrodes

22 and 23 are energized, and the DEP forces in the two directions are superposed, the resultant force is downward, and the root of the DEP force in one direction is 2 times of the root of the DEP force in one direction due to the positions of the 3 electrodes at almost 45 degrees, so that the cell shape is 0.18 to 0.35.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A micro-fluidic chip for multi-angle mechanical measurement of cells is characterized by being formed by packaging a substrate and a cover plate, wherein the cover plate is a PDMS substrate provided with a micro-channel, AB sections and AC sections of the micro-channel are symmetrically arranged relative to an AD section positioned on a central line, AE sections are linear extension sections of the AD section, the AB sections and the AC sections have the same width, the AD sections and the AE sections have the same width and are wider than the AB sections and the AC sections, a first liquid inlet, a second liquid inlet, a third liquid inlet and a liquid outlet are respectively and correspondingly arranged at the B end, the C end, the D end and the E end of the micro-channel, and the angle BAC is 30-60 degrees; the substrate is ITO glass with the size slightly larger than that of the cover plate, electrodes are etched on the ITO layer of the substrate, are positioned in the AE section of the micro-channel and are tightly bonded with the AE section of the micro-channel, and comprise an upper electrode and two lower electrodes which are mutually spaced.

2. The microfluidic chip according to claim 1, wherein the width ratio of the AB segment, the AC segment, the AD segment and the AE segment of the microchannel is 1: 2.0-3.0.

3. The microfluidic chip according to claim 1 or 2, wherein the width of the AB segment and the AC segment of the microchannel is 200 micrometers, and the width of the AD segment and the AE segment is 450 micrometers.

4. The microfluidic chip of claim 1, wherein angle BAC is 60 °.

5. The microfluidic chip according to claim 1, wherein the distance between the upper electrode and the lower electrode is 30-60 microns, and the distance between the two lower electrodes is 15-30 microns.

6. A method for manufacturing a microfluidic chip for multi-angle mechanical measurement of cells is characterized by comprising the following steps:

s1, etching an upper electrode and two lower electrodes which are mutually spaced on an ITO layer of the ITO glass to be used as substrates;

s2, photoetching to obtain an anode membrane, reversing a mold to obtain a PDMS microchannel, wherein an AB section and an AC section of the microchannel are symmetrically arranged relative to an AD section positioned on a central line, an AE section is a linear extension section of the AD section, the AB section and the AC section have the same width, the AD section and the AE section have the same width and are wider than the AB section and the AC section, a first liquid inlet, a second liquid inlet, a third liquid inlet and a third liquid outlet are respectively and correspondingly arranged at the end B, the end C, the end D and the end E of the microchannel, and the angle BAC is 30-60 degrees;

and S3, aligning the ITO glass substrate with the PDMS cover plate through an alignment platform to enable the electrode to be positioned in the AE section of the micro-channel and tightly bonded to obtain the micro-fluidic chip.

7. The method of claim 6, wherein the width ratio of the AB section, the AC section, the AD section and the AE section of the microchannel is 1: 2.0-3.0.

8. The method according to claim 6 or 7, wherein the width of the AB section and the AC section of the microchannel is 200 μm, and the width of the AD section and the AE section is 450 μm.

9. The method of claim 6, wherein angle BAC is 60 °.

10. The method of claim 6, wherein the upper electrode and the lower electrode are spaced apart by 30 to 60 microns, and the spacing between the two lower electrodes is 15 to 30 microns.