CN108160127B - Microfluidic separation chip and manufacturing method thereof - Google Patents
Microfluidic separation chip and manufacturing method thereof Download PDFInfo
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- CN108160127B CN108160127B CN201810092602.2A CN201810092602A CN108160127B CN 108160127 B CN108160127 B CN 108160127B CN 201810092602 A CN201810092602 A CN 201810092602A CN 108160127 B CN108160127 B CN 108160127B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/12—Specific details about manufacturing devices
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Abstract
The invention discloses a microfluidic separation chip and a manufacturing method thereof, and the microfluidic separation chip comprises a PDMS substrate and a PDMS cover plate, wherein the PDMS substrate is provided with a main channel, a separation fulcrum and a separation channel are arranged on the main channel, the PDMS substrate is provided with 1 liquid inlet tank, 8 separation liquid storage tanks and 8 electroosmotic flow control electrodes corresponding to the 8 separation liquid storage tanks, and 1 PDMS insulation with the thickness of 5 micrometers is arranged between the 8 electroosmotic flow control electrodes and the 8 separation liquid storage tanks. And liquid storage tanks and liquid inlet tanks with the same sizes as the 8 separation liquid storage tanks and the liquid inlet tanks on the substrate are arranged at corresponding positions at the bottom of the PDMS cover plate, and glass beads with the diameter of 5 micrometers are respectively placed at positions on the cover plate corresponding to the separation fulcrums on the substrate. The invention has the advantages of low voltage required for dielectric breakdown, high safety and operability of experiment and low experiment cost, and can realize automatic control and separation of samples.
Description
Technical Field
The invention relates to a microfluidic separation chip and a manufacturing method thereof.
Background
The micro-fluidic chip, also called "Lab-on-a-chip", is a chip with a small area, which is manufactured by micro-electromechanical technology to form micro-pump, micro-valve, micro-electrode, micro-filter, micro-reactor and other structural elements, so as to realize the micro-integration of the macroscopic functions of all laboratory units and finally realize the functions of Lab-on-a-chip. Compared with the traditional biochemical sample detection and preparation technology, the microfluidic chip technology has the advantages of less sample loss, quick detection response and simple operation, can shorten the detection time and improve the sensitivity, accuracy and efficiency of detection, is gradually popularized and applied in the field of biochemical detection, and has very important application value.
At present, in the microfluidic chip designed by most scholars, the control of a sample detection product is unidirectional, manual intervention is high, the automation degree is low, and separation and extraction of different products have certain difficulty, so that the application and popularization of the technology are restricted.
According to the research of scientists, the dielectric breakdown of some materials under specific conditions can not cause permanent damage and has recoverability.
Breakdown is classified into thermal breakdown and electrical breakdown, in which electrical breakdown is intrinsic breakdown due to electron instability at room temperature or low temperature.
The factors that cause dielectric breakdown of solid dielectrics under high electric fields are: temperature and field strength reach thermal breakdown or arc discharge causes destruction. The method is carried out in a channel filled with conductive liquid, and breakdown damage caused by arc discharge can be avoided in a sealed environment. In order to avoid damage from thermal breakdown, once the field strength of the intrinsic breakdown is reached, the voltage is no longer increased; the increase of heat is reduced by various means as far as possible, and the damage caused by thermal breakdown is avoided.
The formation of an arc discharge arc is a process in which air-neutral protons (molecules and atoms) are dissociated between the electrodes. In the Klein experiment, voltage is applied to two ends of an electrode to generate current of 10 amperes within microseconds, and the current density can reach 1010A/cm2. Because the experiment is carried out in the channel filled with the conductive liquid, the breakdown damage caused by arc discharge can be avoided in a sealed environment.
The thermal breakdown has a definite critical breakdown field strength, which is in the order of 108V/m or higher, a large amount of heat may cause damage (melting) of the material. In order to avoid damage from thermal breakdown, once the field strength of the intrinsic breakdown is reached, the voltage is no longer increased; the increase of heat is reduced by various means as far as possible, and the damage caused by thermal breakdown is avoided.
Heat Q entering the system from the heat conducting system per unit time through the surface a1Heat Q generated by an internal heat source (electric heating) per unit volume (AL) per unit time2Increase in Heat per volume per time Q3。
The heat conduction system establishes an energy balance equation: q1+Q2＝Q3. (assumed to be in steady state model) in steady state, Q3Is 0, namely the heat generated by the internal heat source is completely conducted out without causing internal energy change.
The equation for thermal conductivity is:
A(m2) -a cross section of a heat conducting cross section, L (m) -a thickness of two heat sources, Δ T (K) -a temperature difference,
heat conducted directly per unit time, thermal conductivity k (W/mK) — the ability of a material to conduct heat.
The simulation data shows that the thickness of the channel wall (used for dielectric breakdown control) of the microfluidic chip is designed to be within 5 microns, so that breakdown damage can be effectively prevented, and the diameter of the corresponding channel is designed to be 10 microns.
The breakdown field strength of PDMS is 140kV/cm, and the required breakdown field strength is lower than that of glass. Pdms (polydimethylsiloxane), polydimethylsiloxane is used in a large number of micro flow channel systems in bio-mems, soft etching (soft lithography) technology. Channels are designed in the silicon plates, then liquid dimethyl siloxane is poured in the silicon plates and heated to harden the dimethyl siloxane. When the dimethylsiloxane is removed, even micro-scale micro-channel design details are printed on the PDMS (polydimethylsiloxane) plate. With this particular silicone rubber sheet, hydrophilic surface modification was performed using a Reactive Ion Etcher (RIE). Once the surface bonds are broken, it is common to place a glass slide over the activated side of the silicone (side traces). Once bonded back to normal, the glass permanently bonds to the PDMS plate, changing the channel originally designed in the silicon plate into a water-tight channel. With the technology, the elements such as micro-channels, micro-mixers, micro-pumps, micro-valves and the like are manufactured at low cost, and the minimum geometric dimension can also reach the nanometer level.
Therefore, in order to improve the separation effect and efficiency of the product in the lab-on-a-chip, from the practical application point of view, the invention designs a digital microfluidic separation chip based on the dielectric breakdown technology and provides a manufacturing method thereof.
Disclosure of Invention
The invention aims to provide a microfluidic separation chip and a manufacturing method thereof, wherein the voltage required for dielectric breakdown is low, the automatic control separation of samples can be realized, the safety and operability of experiments are improved, and the experiment cost is reduced.
The technical solution of the invention is as follows:
a microfluidic separation chip comprises a PDMS (polydimethylsiloxane) substrate and a PDMS cover plate, wherein the PDMS substrate and the PDMS cover plate are cuboids, the bottom of the PDMS substrate is a square with the side length of 6 cm and the height of the PDMS cover plate is 3 mm, and the PDMS substrate and the PDMS cover plate are bonded.
The center of the PDMS substrate is provided with a cylindrical substrate liquid inlet tank, the height of the substrate liquid inlet tank is 10 micrometers, and the diameter of a bottom circle is 3 millimeters.
On the PDMS substrate, a cuboid main channel is arranged in front of the substrate liquid inlet tank and connected with the substrate liquid inlet tank, the main channel is parallel to the bottom surface of the PDMS substrate, the length of the main channel is 1.75 cm, the width of the main channel is 10 microns, and the height of the main channel is 10 microns.
The main channel is provided with 4 separation fulcrums which are sequentially spaced by 0.25 cm, the distance between the last separation fulcrum and the substrate liquid inlet tank is 1 cm, a pair of separation channels which are symmetrical to each other is arranged on the left side and the right side of each separation fulcrum, the separation channels on the two sides of the frontmost separation fulcrum are perpendicular to the main channel, other separation channels are forward and form 60-degree included angles with the main channel, the width of each separation channel is 10 micrometers, and the height of each separation channel is 10 micrometers.
On the PDMS substrate, the left side and the right side of the foremost separation fulcrum on the main channel are transversely and symmetrically provided with 4 cylindrical substrate separation liquid storage tanks with the same size, the substrate separation liquid storage tanks are sequentially connected with the separation channel, a pair of substrate separation liquid storage tanks closest to the main channel are separated by 0.4 cm and are respectively connected with the separation channels on the two sides of the foremost separation fulcrum, the diameter of the bottom circle of each substrate separation liquid storage tank is 2 mm, and the height of each substrate separation liquid storage tank is 10 microns.
8 electroosmotic flow control electrodes opposite to the 8 substrate separation liquid storage tanks are arranged in front of the PDMS substrate, and 1 PDMS insulation with the thickness of 5 micrometers is arranged between the 8 electroosmotic flow control electrodes and the 8 substrate separation liquid storage tanks.
The PDMS cover plate is equipped with 1 cylinder cover plate liquid inlet tank with the base piece liquid inlet tank corresponding position department, and cover plate liquid inlet tank bottom surface circle diameter is 3 millimeters, and the height is 1 millimeter.
PDMS cover plate and 8 substrate separation liquid storage ponds correspond the position department and are equipped with 8 cylindric cover plate separation liquid storage ponds of cylinder, and cover plate separation liquid storage pond bottom surface circle diameter is 2 millimeters, and the height is 1 millimeter.
4 groups of glass beads which are close to each other in the transverse direction are glued on the PDMS cover plate, each group of glass beads consists of 3 glass beads with the diameter of 5 micrometers, and the glass beads in the middle of each group of glass beads correspond to the position of the separation pivot on the PDMS substrate.
Bonding the PDMS substrate and the PDMS cover plate comprises the following steps: the corresponding surfaces of the substrate liquid inlet pool, the separation fulcrum, the substrate separation liquid storage pool and the cover plate liquid inlet pool of the PDMS cover plate, the glass beads and the cover plate separation liquid storage pool are jointed.
A method for manufacturing a microfluidic separation chip comprises the following steps:
a) all patterns of a chip substrate are obtained on the silicon chip by adopting a silicon chip photoetching process, and the silicon chip is etched by utilizing an ICP (Inductively coupled Plasma etching) technology to obtain the structural shapes of a substrate liquid inlet pool, a main channel, a separation channel, a substrate separation liquid storage pool, an insulation and an electroosmosis flow control electrode.
b) The method comprises the following steps of using a silicon chip as a mold, selecting a PDMS material to cast on the silicon chip, and performing mold rotation to obtain a PDMS (polydimethylsiloxane) substrate, wherein the structure of the PDMS substrate is as follows:
the PDMS substrate is a cuboid, the bottom of the PDMS substrate is a square with the side length of 6 cm and the height of 3 mm.
The center of the PDMS substrate is provided with a cylindrical substrate liquid inlet tank, the height of the substrate liquid inlet tank is 10 micrometers, and the diameter of a bottom circle is 3 millimeters.
On the PDMS substrate, a cuboid main channel is arranged in front of the substrate liquid inlet tank and connected with the substrate liquid inlet tank, the main channel is parallel to the bottom surface of the PDMS substrate, the length of the main channel is 1.75 cm, the width of the main channel is 10 microns, and the height of the main channel is 10 microns.
The main channel is provided with 4 separation fulcrums which are sequentially spaced by 0.25 cm, the distance between the last separation fulcrum and the substrate liquid inlet tank is 1 cm, a pair of separation channels which are symmetrical to each other is arranged on the left side and the right side of each separation fulcrum, the separation channels on the two sides of the frontmost separation fulcrum are perpendicular to the main channel, other separation channels are forward and form 60-degree included angles with the main channel, the width of each separation channel is 10 micrometers, and the height of each separation channel is 10 micrometers.
On the PDMS substrate, the left side and the right side of the foremost separation fulcrum on the main channel are transversely and symmetrically provided with 4 cylindrical substrate separation liquid storage tanks with the same size, the substrate separation liquid storage tanks are sequentially connected with the separation channel, a pair of substrate separation liquid storage tanks closest to the main channel are separated by 0.4 cm and are respectively connected with the separation channels on the two sides of the foremost separation fulcrum, the diameter of the bottom circle of each substrate separation liquid storage tank is 2 mm, and the height of each substrate separation liquid storage tank is 10 microns.
8 electroosmotic flow control electrodes opposite to the 8 substrate separation liquid storage tanks are arranged in front of the PDMS substrate, and 1 PDMS insulation with the thickness of 5 micrometers is arranged between the 8 electroosmotic flow control electrodes and the 8 substrate separation liquid storage tanks.
8 electroosmotic flow control electrodes on the PDMS substrate are respectively formed by gluing copper wires with the diameter of 100 micrometers on corresponding positions, and the length of each copper wire protrudes 1 cm from the edge of the PDMS substrate and is used for electrically connecting the chip with the outside.
c) The method comprises the steps of manufacturing a square with the size and the shape being 6 centimeters in length at the bottom which is the same as that of a PDMS substrate, manufacturing a cuboid paper box with the height being 3 millimeters, gluing 8 diameters on the positions corresponding to 8 PDMS substrate separation liquid storage tanks respectively to be 2 millimeters, gluing the solid cylinder model with the height being 1 millimeter, injecting PDMS materials into the paper box, curing, stripping to obtain a PDMS cover plate, arranging 1 cylindrical cover plate liquid storage tank on the positions corresponding to the PDMS substrate separation liquid storage tanks, arranging 3 millimeters in diameter of a bottom circle of the cover plate liquid storage tank, and arranging 1 millimeter in diameter, wherein the positions corresponding to the PDMS cover plate and the 8 substrate separation liquid storage tanks are provided with 8 cylindrical cover plate separation liquid storage tanks, and the diameters of the bottom circle of the cover plate separation liquid storage tank are 2 millimeters and 1 millimeter.
4 groups of glass beads which are close to each other in the transverse direction are glued on the PDMS cover plate, each group of glass beads consists of 3 glass beads with the diameter of 5 micrometers, and the glass beads in the middle of each group of glass beads correspond to the position of the separation pivot on the PDMS substrate.
d) Bonding a PDMS substrate with a PDMS cover: placing a PDMS substrate and a PDMS cover plate at a position 3 cm below a 6-watt low-pressure mercury lamp, irradiating for 3 hours, dripping deionized water on the PDMS substrate and the PDMS cover plate, jointing corresponding surfaces of a substrate liquid inlet pool, a separation fulcrum, a substrate separation liquid storage pool and a cover plate liquid inlet pool, a glass bead and a cover plate separation liquid storage pool of the PDMS cover plate within 1 minute, folding, placing in a drying box for storage at 65 ℃ for 2 hours, taking out, and placing in the air for 24 hours for later use.
Has the advantages that:
the invention has 1 layer thickness 5 micron insulating dam between the electroosmotic flow control electrode and the separation liquid storage tank for isolation, and when separating sample, all channels are filled with conductive liquid, therefore, the voltage applied on the liquid inlet tank can be directly transmitted to the separation liquid storage tank through the conductive liquid, thus forming an electric field between the electroosmotic flow control electrode and the separation liquid storage tank, namely forming an electric field between the electroosmotic flow control electrode and the liquid inlet tank, and the separation of sample can be realized by electroosmotic flow drive formed by the electric field applied between the liquid inlet tank and the control electrode. As the breakdown field intensity of PDMS is 140kV/cm, for a PDMS insulation dam with the width of 5 microns, the voltage required for carrying out dielectric breakdown and forming electroosmotic flow in a channel only needs about 70 volts, which is far lower than the kilovolt voltage required for carrying out electroosmotic flow separation by the traditional method, so that the safety and operability of an experiment are improved, and the experiment cost is reduced. In addition, in order to realize the automatic control of the separation of the sample, glass beads with the diameter of 5 millimeters are respectively arranged at the separation branch points of the main channel, so that the amplification technology of an optical lens and the image acquisition sensing technology can be combined to monitor the types of the samples in the channel in real time, the digital image processing technology is utilized to carry out the characteristic recognition of the images of the samples, the recognized images are compared with the database data stored in a computer to determine the types of the samples, and then according to the requirements of the separation of the samples, a computer outputs a control instruction to an electrode switching electronic control switch to realize the switching control of different sample separation channels and realize the automatic control and separation of the samples.
Drawings
FIG. 1 is a diagram of a microfluidic separation chip;
FIG. 2 is a flow chart of the photolithography and etching processes of a silicon wafer;
FIG. 3 is a flow chart of PDMS chip fabrication.
Detailed Description
The invention will be described in further detail below with reference to the following figures and specific examples:
example 1: a microfluidic separation chip is shown in FIG. 1 and comprises a PDMS (polydimethylsiloxane) substrate and a PDMS cover plate, wherein the PDMS substrate and the PDMS cover plate are cuboids, the bottom of the PDMS substrate is a square with the side length of 6 cm and the height of the PDMS cover plate is 3 mm, and the PDMS substrate and the PDMS cover plate are bonded.
The center of the PDMS substrate is provided with a cylindrical substrate liquid inlet tank, the height of the substrate liquid inlet tank is 10 micrometers, and the diameter of a bottom circle is 3 millimeters.
On the PDMS substrate, a cuboid main channel is arranged in front of the substrate liquid inlet tank and connected with the substrate liquid inlet tank, the main channel is parallel to the bottom surface of the PDMS substrate, the length of the main channel is 1.75 cm, the width of the main channel is 10 microns, and the height of the main channel is 10 microns.
The main channel is provided with 4 separation fulcrums which are sequentially spaced by 0.25 cm, the distance between the last separation fulcrum and the substrate liquid inlet tank is 1 cm, a pair of separation channels which are symmetrical to each other is arranged on the left side and the right side of each separation fulcrum, the separation channels on the two sides of the frontmost separation fulcrum are perpendicular to the main channel, other separation channels are forward and form 60-degree included angles with the main channel, the width of each separation channel is 10 micrometers, and the height of each separation channel is 10 micrometers.
On the PDMS substrate, the left side and the right side of the foremost separation fulcrum on the main channel are transversely and symmetrically provided with 4 cylindrical substrate separation liquid storage tanks with the same size, the substrate separation liquid storage tanks are sequentially connected with the separation channel, a pair of substrate separation liquid storage tanks closest to the main channel are separated by 0.4 cm and are respectively connected with the separation channels on the two sides of the foremost separation fulcrum, the diameter of the bottom circle of each substrate separation liquid storage tank is 2 mm, and the height of each substrate separation liquid storage tank is 10 microns.
8 electroosmotic flow control electrodes opposite to the 8 substrate separation liquid storage tanks are arranged in front of the PDMS substrate, and 1 PDMS insulation with the thickness of 5 micrometers is arranged between the 8 electroosmotic flow control electrodes and the 8 substrate separation liquid storage tanks.
The PDMS cover plate is equipped with 1 cylinder cover plate liquid inlet tank with the base piece liquid inlet tank corresponding position department, and cover plate liquid inlet tank bottom surface circle diameter is 3 millimeters, and the height is 1 millimeter.
PDMS cover plate and 8 substrate separation liquid storage ponds correspond the position department and are equipped with 8 cylindric cover plate separation liquid storage ponds of cylinder, and cover plate separation liquid storage pond bottom surface circle diameter is 2 millimeters, and the height is 1 millimeter.
4 groups of glass beads which are close to each other in the transverse direction are glued on the PDMS cover plate, each group of glass beads consists of 3 glass beads with the diameter of 5 micrometers, and the glass beads in the middle of each group of glass beads correspond to the position of the separation pivot on the PDMS substrate.
Bonding the PDMS substrate and the PDMS cover plate comprises the following steps: the corresponding surfaces of the substrate liquid inlet pool, the separation fulcrum, the substrate separation liquid storage pool and the cover plate liquid inlet pool of the PDMS cover plate, the glass beads and the cover plate separation liquid storage pool are jointed.
A method for manufacturing a microfluidic separation chip comprises the following steps:
a) all patterns of a chip substrate are obtained on the silicon chip by adopting a silicon chip photoetching process, and the silicon chip is etched by utilizing an ICP (Inductively coupled Plasma etching) technology to obtain the structural shapes of a substrate liquid inlet pool, a main channel, a separation channel, a substrate separation liquid storage pool, an insulation and an electroosmosis flow control electrode.
As shown in fig. 2, the process flow of the lithography and etching of the silicon wafer is as follows:
cleaning: and ultrasonically cleaning the silicon wafer for 5 minutes respectively by using acetone, alcohol and deionized water, blow-drying by using nitrogen, and drying on a hot plate at the temperature of 150 ℃ for 2-5 minutes.
Glue homogenizing: the glue is homogenized in two steps, wherein the glue is rotated for 15-20 seconds at 500r/min in the first step, and is rotated for 30-35 seconds at 4000r/min in the second step.
Pre-exposure: the ultraviolet exposure is carried out for 90s,
post-baking: baking the mixture for 120s on a hot plate at 120 ℃,
post exposure: the ultraviolet exposure is carried out for 160s,
and (3) developing: developing in the developing solution for 40-45 s,
sputtering: RF power of 120W and vacuum degree of 4X 10-4Pa, argon pressure of 5mTorr, and substrate cooling mode of water cooling. The sputtering rate is about 16nm/min, and the sputtering time is 12 min. The Cr film thickness is about 100 nm.
Then, the resist was peeled off, and etching was performed by using a built-in Bosch process using an etcher of an Oxford Instruments Plasma-lab system 100. The built-in inductively coupled plasma coupling parameters are shown in the following table:
b) the method comprises the following steps of using a silicon chip as a mold, selecting a PDMS material to cast on the silicon chip, and performing mold rotation to obtain a PDMS (polydimethylsiloxane) substrate, wherein the structure of the PDMS substrate is as follows:
the PDMS substrate is a cuboid, the bottom of the PDMS substrate is a square with the side length of 6 cm and the height of 3 mm.
The center of the PDMS substrate is provided with a cylindrical substrate liquid inlet tank, the height of the substrate liquid inlet tank is 10 micrometers, and the diameter of a bottom circle is 3 millimeters.
On the PDMS substrate, a main channel is arranged in front of the substrate liquid inlet tank and connected with the substrate liquid inlet tank, the main channel is parallel to the bottom surface of the PDMS substrate, the length of the main channel is 1.75 cm, the width of the main channel is 10 microns, and the height of the main channel is 10 microns.
The main channel is provided with 4 separation fulcrums which are sequentially spaced by 0.25 cm, the distance between the last separation fulcrum and the substrate liquid inlet tank is 1 cm, a pair of separation channels which are symmetrical to each other is arranged on the left side and the right side of each separation fulcrum, the separation channels on the two sides of the frontmost separation fulcrum are perpendicular to the main channel, other separation channels are forward and form 60-degree included angles with the main channel, the width of each separation channel is 10 micrometers, and the height of each separation channel is 10 micrometers.
On the PDMS substrate, the left side and the right side of the foremost separation fulcrum on the main channel are transversely and symmetrically provided with 4 cylindrical substrate separation liquid storage tanks with the same size, the substrate separation liquid storage tanks are sequentially connected with the separation channel, a pair of substrate separation liquid storage tanks closest to the main channel are separated by 0.4 cm and are respectively connected with the separation channels on the two sides of the foremost separation fulcrum, the diameter of the bottom circle of each substrate separation liquid storage tank is 2 mm, and the height of each substrate separation liquid storage tank is 10 microns.
8 electroosmotic flow control electrodes opposite to the 8 substrate separation liquid storage tanks are arranged in front of the PDMS substrate, and 1 PDMS insulation with the thickness of 5 micrometers is arranged between the 8 electroosmotic flow control electrodes and the 8 substrate separation liquid storage tanks.
8 electroosmotic flow control electrodes on the PDMS substrate are respectively formed by gluing copper wires with the diameter of 100 micrometers on corresponding positions, and the length of each copper wire protrudes 1 cm from the edge of the PDMS substrate and is used for electrically connecting the chip with the outside.
c) The method comprises the steps of manufacturing a square with the bottom side length being 6 centimeters and the same in size and shape as a PDMS substrate, manufacturing a cuboid paper box with the height being 3 millimeters, gluing and placing the solid cylindrical model with the diameter being 3 millimeters at the position of the cuboid paper box corresponding to a PDMS substrate liquid inlet tank, gluing and placing the solid cylindrical model with the diameter being 1 millimeter at the position of the solid cylindrical model corresponding to 8 PDMS substrate liquid inlet tanks, respectively placing the solid cylindrical model with the diameter being 2 millimeters at the position of the solid cylindrical model corresponding to 8 PDMS substrate liquid inlet tanks, injecting PDMS materials into the paper box, curing, stripping to obtain a PDMS cover plate, arranging 1 cylindrical cover plate liquid inlet tank at the position of the PDMS cover plate corresponding to the substrate liquid inlet tank, arranging 8 cylindrical cover plate separation liquid storage tanks at the position of the PDMS cover plate corresponding to the substrate liquid inlet tank, arranging the cylindrical cover plate separation liquid storage tanks at the position of the PDMS cover plate corresponding to the substrate separation liquid storage tanks, and.
4 groups of glass beads which are close to each other in the transverse direction are glued on the PDMS cover plate, each group of glass beads consists of 3 glass beads with the diameter of 5 micrometers, and the glass beads in the middle of each group of glass beads correspond to the position of the separation pivot on the PDMS substrate.
d) Bonding a PDMS substrate with a PDMS cover: placing a PDMS substrate and a PDMS cover plate at a position 3 cm below a 6-watt low-pressure mercury lamp, irradiating for 3 hours, dripping deionized water on the PDMS substrate and the PDMS cover plate, jointing corresponding surfaces of a substrate liquid inlet pool, a separation fulcrum, a substrate separation liquid storage pool and a cover plate liquid inlet pool, a glass bead and a cover plate separation liquid storage pool of the PDMS cover plate within 1 minute, folding, placing in a drying box for storage at 65 ℃ for 2 hours, taking out, and placing in the air for 24 hours for later use.
The process of making PDMS chip is shown in FIG. 3.
According to the invention, copper wires with the diameter of 100 micrometers are glued at the positions of 8 electroosmotic flow control electrodes, the length of each copper wire protrudes 1 cm from the edge of a PDMS substrate, and the copper wires are connected to an output pin of an external multi-channel analog control switch chip CD 4051.
Glass beads with the diameter of 5 millimeters are respectively arranged at the separation branch points of the main channel, so that the type of a sample in the channel can be monitored in real time by combining an optical lens amplification technology and an image acquisition sensing technology, the characteristic identification of the sample image is carried out by utilizing a digital image processing technology, the identified image is compared with database data stored in a computer to determine the type of the sample, then a control instruction is output to an external multi-channel analog control switch chip CD4051 by the computer according to the requirement of sample separation, the switching control of different sample separation channels is realized by utilizing three input channel selection ends ABC of the external multi-channel analog control switch chip CD4051, and the automatic control separation of the sample is realized.
The invention has 1 layer thickness 5 micron insulating dam between the electroosmotic flow control electrode and the separation liquid storage tank for isolation, and when separating sample, all channels are filled with conductive liquid, therefore, the voltage applied on the liquid inlet tank can be directly transmitted to the separation liquid storage tank through the conductive liquid, thus forming an electric field between the electroosmotic flow control electrode and the separation liquid storage tank, namely forming an electric field between the electroosmotic flow control electrode and the liquid inlet tank, and the separation of sample can be realized by electroosmotic flow drive formed by the electric field applied between the liquid inlet tank and the control electrode. As the breakdown field intensity of PDMS is 140kV/cm, for a PDMS insulation dam with the width of 5 microns, the voltage required for carrying out dielectric breakdown and forming electroosmotic flow in a channel only needs about 70 volts, which is far lower than the kilovolt voltage required for carrying out electroosmotic flow separation by the traditional method, so that the safety and operability of an experiment are improved, and the experiment cost is reduced.
Claims (4)
1. A microfluidic separation chip is characterized by comprising a PDMS (polydimethylsiloxane) substrate and a PDMS cover plate, wherein the PDMS substrate and the PDMS cover plate are cuboids, the bottom of the PDMS substrate is a square with the side length of 6 cm and the height of the PDMS cover plate is 3 mm, the PDMS substrate and the PDMS cover plate are bonded,
a cylindrical substrate liquid inlet tank is arranged at the center of the PDMS substrate, the height of the substrate liquid inlet tank is 10 micrometers, the diameter of a bottom circle is 3 millimeters,
on the PDMS substrate, a cuboid main channel is arranged in front of the substrate liquid inlet tank and connected with the substrate liquid inlet tank, the main channel is parallel to the bottom surface of the PDMS substrate, the length of the main channel is 1.75 cm, the width of the main channel is 10 microns, the height of the main channel is 10 microns,
the main channel is provided with 4 separation fulcrums which are sequentially spaced by 0.25 cm, the distance between the last separation fulcrum and the substrate liquid inlet pool is 1 cm, a pair of separation channels which are symmetrical to each other are arranged on the left side and the right side of each separation fulcrum, the separation channels on the two sides of the frontmost separation fulcrum are perpendicular to the main channel, other separation channels are forward and form 60-degree included angles with the main channel, the width of each separation channel is 10 micrometers, the height of each separation channel is 10 micrometers,
on the PDMS substrate, the left side and the right side of the foremost separation pivot on the main channel are respectively and transversely symmetrically provided with 4 cylindrical substrate separation liquid storage tanks with equal size, the substrate separation liquid storage tanks are sequentially connected with the separation channel, a pair of substrate separation liquid storage tanks closest to the main channel is separated by 0.4 cm and is respectively connected with the separation channels on the two sides of the foremost separation pivot, the diameter of the bottom circle of each substrate separation liquid storage tank is 2 mm, and the height of each substrate separation liquid storage tank is 10 microns,
8 electroosmotic flow control electrodes opposite to the 8 substrate separation liquid storage tanks are arranged in front of the PDMS substrate, a PDMS insulating dam with the thickness of 5 microns is arranged between the 8 electroosmotic flow control electrodes and the 8 substrate separation liquid storage tanks,
the position of the PDMS cover plate corresponding to the substrate liquid inlet tank is provided with 1 cylindrical cover plate liquid inlet tank, the diameter of the circle of the bottom surface of the cover plate liquid inlet tank is 3 mm, the height of the circle is 1 mm,
the PDMS cover plate is equipped with 8 cylindric cover plate separation liquid storage tanks with 8 substrate separation liquid storage tanks corresponding position department, cover plate separation liquid storage tank bottom surface circle diameter is 2 millimeters, and the height is 1 millimeter.
2. The microfluidic separation chip of claim 1, wherein 4 sets of glass beads are glued on the PDMS cover plate and closely arranged in the transverse direction, each set of glass beads is composed of 3 glass beads with the diameter of 5 microns, and the glass bead in the middle of each set of glass beads corresponds to the position of the separation pivot on the PDMS substrate.
3. The microfluidic separation chip according to claim 2, wherein the bonding of the PDMS substrate and the PDMS cover comprises the following steps: the corresponding surfaces of the substrate liquid inlet tank, the separation fulcrum, the substrate separation liquid storage tank and the cover plate liquid inlet tank of the PDMS cover plate, the glass beads and the cover plate separation liquid storage tank are combined together.
4. The manufacturing method of the microfluidic separation chip is characterized by comprising the following steps of:
a) obtaining all patterns of a chip substrate on a silicon chip by adopting a silicon chip photoetching process, and etching the silicon chip by utilizing an ICP (Inductively coupled Plasma etching) technology to obtain the structural shapes of a substrate liquid inlet pool, a main channel, a separation channel, a substrate separation liquid storage pool, an insulation dam and an electroosmotic flow control electrode;
b) the method comprises the following steps of using a silicon chip as a mold, selecting a PDMS material to cast on the silicon chip, and performing mold rotation to obtain a PDMS (polydimethylsiloxane) substrate, wherein the structure of the PDMS substrate is as follows:
the PDMS substrate is a cuboid, the bottom of the PDMS substrate is a square with the side length of 6 cm and the height of 3 mm,
a cylindrical substrate liquid inlet tank is arranged at the center of the PDMS substrate, the height of the substrate liquid inlet tank is 10 micrometers, the diameter of a bottom circle is 3 millimeters,
on the PDMS substrate, a cuboid main channel is arranged in front of the substrate liquid inlet tank and connected with the substrate liquid inlet tank, the main channel is parallel to the bottom surface of the PDMS substrate, the length of the main channel is 1.75 cm, the width of the main channel is 10 microns, the height of the main channel is 10 microns,
the main channel is provided with 4 separation fulcrums which are sequentially spaced by 0.25 cm, the distance between the last separation fulcrum and the substrate liquid inlet pool is 1 cm, a pair of separation channels which are symmetrical to each other are arranged on the left side and the right side of each separation fulcrum, the separation channels on the two sides of the frontmost separation fulcrum are perpendicular to the main channel, other separation channels are forward and form 60-degree included angles with the main channel, the width of each separation channel is 10 micrometers, the height of each separation channel is 10 micrometers,
on the PDMS substrate, the left side and the right side of the foremost separation pivot on the main channel are respectively and transversely symmetrically provided with 4 cylindrical substrate separation liquid storage tanks with equal size, the substrate separation liquid storage tanks are sequentially connected with the separation channel, a pair of substrate separation liquid storage tanks closest to the main channel is separated by 0.4 cm and is respectively connected with the separation channels on the two sides of the foremost separation pivot, the diameter of the bottom circle of each substrate separation liquid storage tank is 2 mm, and the height of each substrate separation liquid storage tank is 10 microns,
8 electroosmotic flow control electrodes opposite to the 8 substrate separation liquid storage tanks are arranged in front of the PDMS substrate, a PDMS insulating dam with the thickness of 5 microns is arranged between the 8 electroosmotic flow control electrodes and the 8 substrate separation liquid storage tanks,
8 electroosmotic flow control electrodes on the PDMS substrate are respectively formed by gluing copper wires with the diameter of 100 micrometers on corresponding positions, and the length of each copper wire protrudes 1 cm from the edge of the PDMS substrate and is used for electrically connecting the chip with the outside;
c) manufacturing a rectangular paper box with the same size and shape as the PDMS substrate, wherein the bottom side length of the rectangular paper box is 6 cm, the height of the rectangular paper box is 3 mm, a solid cylindrical model with the diameter of 3 mm and the height of 1 mm is glued on the position of the rectangular paper box corresponding to the PDMS substrate liquid inlet pool, 8 solid cylindrical models with the diameter of 2 mm and the height of 1 mm are glued on the position of the rectangular paper box corresponding to 8 separated liquid storage pools of the PDMS substrate, PDMS material is selected and injected into the paper box, cured and peeled off to obtain a PDMS cover plate, 1 cylindrical cover plate liquid inlet pool is arranged at the position of the PDMS cover plate corresponding to the substrate liquid inlet pool, the diameter of the bottom circle of the cover plate liquid inlet pool is 3 mm and the height of 1 mm, 8 cylindrical cover plate separated liquid storage pools are arranged at the positions of the PDMS cover plate corresponding to the 8 substrate separated liquid storage pools, the diameter of the bottom circle of the cover plate separated liquid storage pool is,
4 groups of glass beads which are close to each other in the transverse direction are glued on the PDMS cover plate, each group of glass beads consists of 3 glass beads with the diameter of 5 micrometers, and the glass beads in the middle of each group of glass beads correspond to the position of a separation fulcrum on the PDMS substrate;
d) bonding a PDMS substrate with a PDMS cover: placing a PDMS substrate and a PDMS cover plate at a position 3 cm below a 6-watt low-pressure mercury lamp, irradiating for 3 hours, dripping deionized water on the PDMS substrate and the PDMS cover plate, jointing corresponding surfaces of a substrate liquid inlet pool, a separation fulcrum, a substrate separation liquid storage pool and a cover plate liquid inlet pool, a glass bead and a cover plate separation liquid storage pool of the PDMS base plate within 1 minute, folding, placing in a drying box for storage at 65 ℃ for 2 hours, taking out, and placing in the air for 24 hours for later use.
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