CN114887673B - Integrated flow channel digital micro-fluidic chip and preparation method and application thereof - Google Patents

Integrated flow channel digital micro-fluidic chip and preparation method and application thereof Download PDF

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CN114887673B
CN114887673B CN202210446632.5A CN202210446632A CN114887673B CN 114887673 B CN114887673 B CN 114887673B CN 202210446632 A CN202210446632 A CN 202210446632A CN 114887673 B CN114887673 B CN 114887673B
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张涛
何宇
陆泽凡
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Zhejiang University ZJU
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Abstract

The invention discloses an integrated flow channel digital micro-fluidic chip and a preparation method and application thereof. The integrated runner digital microfluidic chip comprises an electrode pattern layer, a dielectric layer covered on the electrode pattern layer and an upper polar plate with a channel structure, wherein the upper polar plate is sealed with the dielectric layer; the upper polar plate with the channel structure comprises a supporting layer, a middle conductive layer and a channel structure layer from top to bottom, wherein the middle conductive layer can be connected with an external electrode. The invention discloses an integrated flow channel digital microfluidic chip which is integrated and sealed, and the chip has the capability of controlling continuous fluid and discrete liquid drops simultaneously by organically integrating a flow channel structure of flow channel microfluidic and an electrode pattern of digital microfluidic; the chip is easy to be connected with an external pipeline, so that continuous sample inlet and outlet fluid control is facilitated; the upper polar plate of the chip contains a conductive layer, so that a bipolar plate digital micro-fluidic chip structure is formed, and the bipolar plate digital micro-fluidic chip structure has all functions of droplet generation, splitting, movement and fusion.

Description

Integrated flow channel digital micro-fluidic chip and preparation method and application thereof
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to an integrated flow channel digital microfluidic chip and a preparation method and application thereof.
Background
The microfluidic chip has the advantages of small sample consumption, high functional integration level, easiness in automatic control and the like, and has important value in the fields of biology, medicine, chemistry, materials, machinery, electronics and the like. According to the control form of the fluid, the microfluidic chip can be mainly divided into two types of flow channel type microfluidic and digital microfluidic. The flow channel type micro-flow control mainly controls the movement of fluid in a closed flow channel through the structural design of the channel, has the advantages of simple fluid driving, high flux, high speed and the like, but the problems of difficult accurate fluid control, low automation degree and the like still exist at present. Compared with the prior art, the digital microfluidic control utilizes the dielectric wetting effect, and changes the solid-liquid surface tension of the chip dielectric layer and the liquid drops above the chip dielectric layer by controlling the voltage applied on the chip electrode array, thereby precisely controlling the movement of the discrete liquid drops, and having the advantages of simple liquid drop control, high automation degree and the like. However, there are also a number of problems with current digital microfluidic chips: 1) The chip packaging process is rough, and after the chip packaging process is usually carried out by using adhesive tape or glue for heightening, an upper polar plate is covered on the upper part of the electrode array simply, so that a sealed chip device cannot be formed, and leakage and cross contamination are easy to occur; 2) The chip is difficult to combine with an external interface, continuous sample inlet and outlet fluid control cannot be realized, and integration of more functional units is seriously hindered; 3) The channel structure design of the chip is limited, i.e. it is difficult to make fluid channels or microstructures with different functions over the electrode array, and thus it is difficult to achieve more complex, accurate fluid manipulation. These problems have greatly limited the further development and application of digital microfluidic technology.
In recent years, the integration of flow channel microfluidics and digital microfluidics has attracted a great deal of attention from researchers, which is expected to realize the complementary advantages of the two and break the respective limitations. However, the current integration schemes are more in solving the interface problem of the two, for example, connecting the two types of chips by using a capillary, or simply connecting the flow channel microfluidic chip at the front end or the back end of the digital microfluidic chip. However, in these integration schemes, the two types of chips are still separate and independent and thus are not truly integrated, and the digital microfluidic chip portion is still formed into a bipolar plate structure by simply capping conductive glass, and thus cannot be formed into a fully enclosed chip. In addition, the fluid or liquid drop in the chip can only be transported in one direction, namely can only enter the electrode area of the digital microfluidic chip from the channel of the flow channel type microfluidic chip, or can only enter the channel of the flow channel type microfluidic chip from the electrode area of the digital microfluidic chip. Although researchers have recently realized orthogonal integration of flow channel type microfluidic and digital microfluidic by sealing a polydimethylsiloxane chip with an optical adhesive dielectric layer, the integrated chip is only a monopole plate digital microfluidic structure and lacks droplet generation and splitting functions because of the difficulty in introducing a grounded upper plate. In addition, the surface hydrophobic modification process of the chip is complex, and the stability is poor.
In general, integration of flow channel type micro-fluidic and digital micro-fluidic is a necessary trend of current micro-fluidic technology development, and is expected to solve various problems of micro-fluidic precision, intelligent control, multifunctional integration and the like, but the current integrated chip still has many defects, including: 1) The two types of chips are mutually independent, and are not really integrated chips; 2) The digital microfluidic chip is simple in package, poor in sealing performance and easy to leak and cross-pollute; 3) The liquid can be transported only in one direction, which is not beneficial to complex and continuous fluid control; 4) Single-plate digital microfluidic structures lack droplet generation and splitting capabilities. Therefore, it is needed to develop an integrated flow channel digital microfluidic chip that is integrated, closed, and has a complete droplet manipulation function, so as to solve the above problems.
Disclosure of Invention
In order to solve the problems, the invention discloses an integrated flow channel digital micro-fluidic chip, which is formed by organically integrating a channel structure and an electrode pattern, can control continuous fluid and accurately control discrete liquid drops; simultaneously, the device is easy to combine with an external interface, thereby realizing continuous sample inlet and outlet fluid control and connection with an upstream functional module and a downstream functional module; in addition, the chip manufacturing process is simple, and a conductive layer can be conveniently embedded between the channel structure layer and the supporting layer to form a bipolar plate digital microfluidic structure, so that all basic functions of droplet generation, splitting, movement and fusion are realized.
The specific technical scheme is as follows:
an integrated flow channel digital micro-fluidic chip comprises an electrode pattern layer, a dielectric layer covered on the electrode pattern layer and an upper polar plate with a channel structure, wherein the upper polar plate is sealed with the dielectric layer;
the upper polar plate with the channel structure comprises a supporting layer, a middle conductive layer and a channel structure layer from top to bottom, and the middle conductive layer can be connected with an external electrode.
The integrated flow channel digital micro-fluidic chip disclosed by the invention can be used for processing various channel structures on an upper polar plate with a channel structure, including but not limited to a fluid channel, a micro-column, a micro-well, a cavity and the like. And the upper polar plate with the channel structure is tightly sealed with the dielectric layer above the electrode pattern layer to form a closed chip; under the supporting effect of the supporting layer of the upper electrode plate with the channel structure, the chip is easy to connect with an external pipeline, thereby being beneficial to continuous and bidirectional fluid control; meanwhile, as the middle conducting layer is introduced between the channel structure layer of the upper polar plate with the channel structure and the supporting layer, the integrated flow channel digital micro-fluidic chip becomes a bipolar plate structure, so that the integrated flow channel digital micro-fluidic chip has all control functions of droplet generation, splitting, movement and fusion.
The channel structure of the channel structure layer is positioned above the electrode pattern of the electrode pattern layer or selectively increased above the non-electrode pattern region of the electrode pattern layer.
When the channel structure of the channel structure layer is positioned above the electrode pattern of the electrode pattern layer and digital microfluidic droplet control is involved, the thickness of the channel structure layer is set to be H, and the height of the channel structure is set to be H, wherein H is selected from 20-500 mu m, and 0 < (H-H) is less than or equal to 50 mu m.
When the channel structure is located above the electrode pattern of the electrode pattern layer and digital microfluidic droplet manipulation is involved, it is necessary to control the height H of the channel structure and the difference between the thicknesses H and H of the channel structure layer at the same time.
Experiments show that when h is selected from 20-500 mu m, the liquid drop driving performance is better. If h is too small, the deformation of the droplet due to the change in contact angle is reduced, resulting in difficulty in driving the droplet; if h is too large, on the one hand, the droplets are significantly affected by gravity, and on the other hand, if larger than the droplet diameter, the droplets are difficult to contact with the upper and lower surfaces at the same time, resulting in difficulty in driving the droplets.
Experiments show that the channel structure layer material with proper thickness (H-H) is needed to exist between the middle conductive layer and the liquid drop in the channel, preferably 0 < (H-H) is less than or equal to 50 mu m, and if the (H-H) is too large, the impedance between the liquid drop and the middle conductive layer caused by the channel structure layer material is larger, so that the driving performance of the liquid drop is poor, and even the liquid drop splitting function is lost. Further preferably, (H-H) =20 μm.
When the channel structure is located above the non-electrode pattern region or above the electrode pattern region where the manipulation of the digital microfluidic droplet is not involved, the height of the channel structure and the thickness of the channel structure layer are not limited, since the region does not involve the function of driving the droplet with an electrode in digital microfluidic.
When digital microfluidic droplet generation and splitting are not involved, an intermediate conductive layer may not be disposed above the channel structure, but droplet manipulation is relatively difficult at this time, and a larger driving voltage is usually required or a ground control function is added to the electrodes in the electrode pattern.
The material of the channel structure layer is one or more selected from polydimethylsiloxane, a photo-curing material and a thermoplastic material.
In the invention, the following components are added:
the photo-curing material is selected from monomers containing photo-curing functional groups and/or oligomers containing photo-curing functional groups, wherein the photo-curing functional groups are selected from one or more of acrylate functional groups, methacrylate functional groups, mercapto functional groups, alkenyl functional groups, vinyl ether functional groups and epoxy functional groups.
The hydrophobic light-cured material is a light-cured material with good hydrophobic property, and is selected from one or more of 1H, 2H-perfluoro decyl acrylate, 2- (perfluoro octyl) ethyl methacrylate, 1H, 2H-perfluoro decyl mercaptan, allyl 1H, 1H-perfluoro octyl ether, polysiloxane acrylate oligomer, perfluoro polyether acrylate oligomer, polysiloxane methacrylate oligomer, perfluoro polyether methacrylate oligomer, mercapto polysiloxane oligomer, mercapto perfluoro polyether oligomer, alkenyl polysiloxane oligomer, alkenyl perfluoro polyether oligomer, epoxy polysiloxane oligomer and epoxy perfluoro polyether oligomer.
The thermoplastic material is selected from polymethyl methacrylate, polystyrene, polycarbonate, and the like.
The channel structure layer is required to have a hydrophobic surface, and when the material adopted for preparing the channel structure layer is a non-hydrophobic material, the channel structure layer can be subjected to surface treatment by a hydrophobic modification reagent so that the surface of the channel structure layer has hydrophobicity; alternatively, the channel structure layer is directly prepared from a material having hydrophobicity.
Preferably, the material of the channel structure layer is selected from photo-curing materials, and the channel structure layer with a hydrophobic surface can be directly formed by utilizing the hydrophobic photo-curing materials; or modifying the channel structure layer into a hydrophobic surface by using a hydrophobic light modifying reagent.
In the invention, the following components are added:
the hydrophobic modification reagent is one or more selected from Teflon, fluorine-containing silane coupling agent and hydrophobic light modification reagent;
the hydrophobic light modifying reagent is selected from one or more of 1H, 2H-perfluoro decyl acrylate, 2- (perfluoro octyl) ethyl methacrylate, 1H, 2H-perfluoro decyl mercaptan and allyl 1H, 1H-perfluoro octyl ether.
Preferably, the upper polar plate with the channel structure is also provided with a sample inlet and a sample outlet for connecting with an external interface; under the supporting effect of the supporting layer, the punching and cutting of the upper polar plate and the connection with an external pipeline are very easy.
The middle conductive layer can be connected with an external grounding electrode, so that the digital microfluidic chip forms a bipolar plate structure, and further has all basic functions of droplet generation, movement, fusion and splitting.
The middle conductive layer is selected from a thin film with a conductive material coated on the surface or a thin layer formed by processing the conductive material;
the conductive material is selected from one or more of conductive oxide (such as ITO, etc.), conductive metal (such as Ag, etc.), conductive polymer (such as PEDOT, etc.), and graphene;
the material of the film is selected from polyester, polyimide, polyethylene terephthalate and the like.
Preferably, the intermediate conductive layer has good transparency for ease of viewing and optical detection.
Preferably, the thickness of the conductive material in the middle conductive layer is 50-300 nm, and the sheet resistance of the formed conductive surface is 5-500 ohms; further preferably, the thickness is 100 to 200nm and the sheet resistance is 5 to 150 ohms.
The material of the supporting layer is one or more selected from polydimethylsiloxane, an elastic supporting body, plastics and glass;
the preparation process of the elastic support body can refer to patent application (a photo-curing micro-fluidic chip based on the elastic support body, and a preparation method and application thereof, application number 202110161473. X).
Preferably:
when the material of the channel structure layer is polydimethylsiloxane, the material of the supporting layer is polydimethylsiloxane, and the two layers can be connected through heat curing or after plasma treatment;
when the material of the channel structure layer is a light-cured material, the material of the supporting layer is selected from an elastic supporting body or glass modified by light-cured functional groups, and the supporting layer and the glass can be connected through ultraviolet photography;
when the material of the channel structure layer is thermoplastic, the material of the supporting layer is selected from plastics, and the two materials can be connected in a thermocompression bonding mode.
The electrode pattern layer is an electrode pattern formed by a plurality of electrodes processed on the substrate, and is connected with an external control circuit;
the substrate is selected from glass, silicon wafer, printed circuit board, plastic board or rubber board;
the material of the plurality of electrodes is one or more selected from conductive oxides (such as ITO, etc.), conductive metals (such as Cu, cr, ag, etc.), conductive polymers (such as PEDOT, etc.), and graphene.
The dielectric layer needs to have hydrophobicity, so that the dielectric layer is a thin layer of hydrophobic dielectric layer material or a composite hydrophobic dielectric layer prepared by using a photo-curing dielectric layer material;
Preferably, the relative dielectric constants of the dielectric layer material and the photo-curing dielectric layer material are all selected from 3-10; the thickness of the dielectric layer is selected from 1-50 mu m. Experiments show that when the dielectric constant and the thickness of the dielectric layer are within the defined parameter range, the chip can be ensured to have good droplet driving performance.
The dielectric layer material is selected from one or more of photoresist, parylene and photo-curing dielectric layer material;
the photocuring medium layer material is a photocuring material with high dielectric constant and is selected from one or more of pentaerythritol tetra (3-mercaptopropionate), tris [2- (3-mercaptopropionic acid) ethyl ] isocyanurate, trimethylolpropane tris (3-mercaptopropionate), pentaerythritol tetra (2-mercaptoacetate), 1,3, 5-triallyl-1, 3, 5-triazine-2, 4,6 (1H, 3H, 5H) -trione and trimethylolpropane diallyl ether;
the photocuring material also comprises a photoinitiator which is one or more selected from benzil compounds, alkyl benzene ketone compounds and acyl phosphorus oxides.
When the dielectric layer material is selected from photoresist and parylene, the dielectric layer material can be directly prepared into a thin layer, and then the thin layer is subjected to surface treatment by using a hydrophobic modification reagent to obtain hydrophobicity.
When the dielectric layer material is selected from the photo-curing dielectric layer materials, one form is to directly prepare the dielectric layer material into a thin layer, and then use a hydrophobic light modification reagent to carry out surface treatment on the thin layer to obtain hydrophobicity; the other form is to prepare a composite hydrophobic medium layer by taking the composite hydrophobic medium layer as a raw material, and the composite hydrophobic medium layer is specifically processed according to one of the following methods:
firstly) forming a light-cured dielectric layer material into a thin layer, curing the light-cured dielectric layer material under the action of light irradiation, and then superposing and curing a hydrophobic light-cured material thin layer on the thin layer to form a composite hydrophobic dielectric layer;
and secondly), uniformly mixing the light-cured dielectric layer material and the hydrophobic light-cured material to form a thin layer, and curing the thin layer to form the composite hydrophobic dielectric layer under the action of light irradiation.
Preferably, the thickness of the formed composite hydrophobic medium layer is selected from 1-50 μm; further, in method one), the thickness of the thin layer of hydrophobic photocurable material is less than 5 μm.
The invention also discloses a preparation method of the integrated flow channel digital micro-fluidic chip, which comprises the following steps: processing an upper polar plate with a channel structure, processing a dielectric layer, processing an electrode pattern layer and sealing a chip, wherein the upper polar plate with the channel structure is processed according to one of the following methods:
A) Placing a curable liquid material on a die, forming a film by a spin coating or knife coating method, then covering the middle conductive layer and the supporting layer, or directly covering the middle conductive layer and the supporting layer on the liquid material to form a film, and curing to form an upper polar plate with a channel structure;
b) Placing a curable liquid material on a die, forming a film by a spin coating or knife coating method, curing to form a channel structure layer, and then connecting the middle conductive layer and the supporting layer to form an upper polar plate with a channel structure;
c) And processing a channel structure on the solid film by a hot pressing method or a laser etching method to form a channel structure layer, and connecting the channel structure layer with the middle conductive layer and the supporting layer to form an upper polar plate with the channel structure.
The electrode pattern layer can be processed by a processing mode common in the field, such as mask lithography, wet etching, laser etching, electronic printing, ink-jet printing, 3D printing and the like.
And processing the dielectric layer, directly coating the material for preparing the dielectric layer on the upper surface of the electrode pattern layer, and closely adsorbing the dielectric layer and the electrode pattern layer by a heating or photo-curing mode.
The chip sealing can be realized by plasma treatment, ultraviolet irradiation, hot pressing and other modes.
Preferably:
when the material of the channel structure layer is polydimethylsiloxane, the material of the medium layer is photoresist, the two materials are sealed by plasma treatment, and then a hydrophobic modification reagent such as Teflon or fluorine-containing silane coupling agent is introduced for surface treatment, so that the channel structure layer and the medium layer have a hydrophobic effect;
when the material of the channel structure layer is a photocuring material, the material of the medium layer is a photocuring medium layer material, the photocuring medium layer material and the channel structure layer are sealed under ultraviolet irradiation, and then a hydrophobic modification reagent Teflon is introduced for surface treatment, or a hydrophobic light modification reagent is introduced and more stable hydrophobic modification is carried out under ultraviolet irradiation, so that the channel structure layer and the medium layer have a hydrophobic effect;
when the channel structure layer is made of thermoplastic materials, the medium layer is made of parylene, and the parylene are sealed by hot pressing, and then a hydrophobic modification reagent Teflon is introduced for surface treatment, so that the channel structure layer and the medium layer have a hydrophobic effect;
further preferred is:
the channel structure layer is made of a hydrophobic photo-curing material, the medium layer is a composite hydrophobic medium layer, and the two layers are sealed under ultraviolet irradiation, and as the channel surface has hydrophobic characteristics, a durable and stable hydrophobic surface can be obtained without hydrophobic treatment.
More preferably, the channel structure layer is made of perfluoropolyether acrylate, and the medium layer is a composite hydrophobic medium layer formed by compounding thiol-ene mixture and perfluoropolyether acrylate as raw materials.
The integrated flow channel digital microfluidic chip disclosed by the invention can control continuous fluid, can accurately control discrete liquid drops, and is beneficial to integration of various complex functions, so that the integrated flow channel digital microfluidic chip has greater advantages and better application prospects in the fields of digital microfluidic, liquid drop microfluidic, biochemical reaction, biomedical research and the like.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses an integrated flow channel digital micro-fluidic chip, wherein an upper polar plate with a channel structure in the chip is tightly sealed with a dielectric layer to form a fully-closed structure, so that the risks of liquid leakage and cross contamination in the experimental process can be effectively avoided; by organically integrating various channel structures and electrode patterns on one chip, the integrated chip has the capability of controlling continuous fluid and discrete liquid drops, is an integrated chip in the true sense, and has wide application prospects in the fields of digital microfluidics, liquid drop microfluidics, biochemical reactions, biomedical research and the like.
The invention also discloses a preparation method of the integrated flow channel digital micro-fluidic chip, which can conveniently embed an intermediate conducting layer between the channel structure layer and the supporting layer in the process of processing the upper polar plate with the channel structure, so that the integrated flow channel digital micro-fluidic chip becomes a bipolar plate structure, thereby having all control functions of droplet generation, splitting, movement and fusion; meanwhile, under the supporting effect of the supporting layer, the chip is very simple and convenient to connect with an external pipeline, so that continuous sample inlet and outlet fluid control and connection with an upstream functional module and a downstream functional module can be performed.
The integrated flow channel digital micro-fluidic chip disclosed by the invention can be prepared from conventional materials such as polydimethylsiloxane, thermoplastic plastics and the like, and can also be prepared from a photo-curing material, wherein an elastic support body is preferably used as a support layer, and a hydrophobic photo-curing material is preferably used as a channel structure layer, so that the processing process of the chip is simple and quick, and hydrophobic treatment is not required; aiming at the dielectric layer, the invention also discloses a composite hydrophobic dielectric layer which has proper dielectric constant and stable hydrophobic characteristic, is matched with the channel structure layer, and can be rapidly and tightly sealed under ultraviolet irradiation, thereby forming a closed integrated flow channel digital micro-fluidic chip.
Drawings
Fig. 1 is a schematic structural diagram of an integrated flow channel digital microfluidic chip according to the present invention, in which:
1-an upper polar plate with a channel structure, 11-a supporting layer, 12-an intermediate conducting layer, 13-a channel structure layer, 14-a sample inlet, 15-a sample outlet, 2-a medium layer, 3-an electrode pattern layer and 31-an electrode;
fig. 2 is a schematic diagram of a process flow of an upper plate with a channel structure, in which:
11-supporting layer, 12-middle conducting layer, 13-channel structure layer, 14-sample inlet, 15-sample outlet, 16-solidifiable liquid material, 17-solid film and 6-mould;
fig. 3 is a mask pattern for electrode pattern layer processing (a pattern), a mask for channel structure layer mold processing (B pattern), and an effect pattern of electrode pattern layer and upper plate with channel structure processed according to two masks (C pattern) in example 1;
FIG. 4 is a photomicrograph of the control of a continuous fluid using the integrated fluidic channel digital microfluidic chip prepared in example 1; (a) For fluid entering the electrode pattern area from the channel, (b) for fluid entering the channel from the electrode pattern area, wherein the white curve represents the contour of the fluid in the chip;
FIG. 5 is a photomicrograph of manipulation of discrete droplets using the integrated fluidic channel digital microfluidic chip prepared in example 4; (a) is a droplet splitting process, (b) is a droplet fusion process, and (c) is a droplet moving process, wherein the droplet generation process is similar to the droplet splitting process, a white curve in the figure represents the outline of a droplet in a chip, and a white arrow represents the movement direction of the droplet;
Fig. 6 is a mask pattern for the mold processing of the channel structure layer in example 5, in which:
1-digital microfluidic nucleic acid extraction region, 11-sample inlet, 12-nucleic acid extraction chamber, 13-nucleic acid solution sample outlet channel, 2-digital nucleic acid detection region, 21-sample distribution channel, 22-oil phase channel, 23-nucleic acid amplification region, 24-sample outlet;
the mold processing adopts a double-layer structure to obtain channels with different depths, and corresponding masks are respectively shown in fig. 6A and 6B;
fig. 7 is a mask pattern for processing an electrode pattern layer in example 5, in which:
1-an external electrode, 2-a connecting wire and 3-an electrode pattern;
FIG. 8 is a photomicrograph of droplet generation in an integrated fluidic digital chip prepared in example 5, (A) water-in-oil droplets generated at a T-shaped droplet generation structure, (B) a plurality of uniform and identical individual droplets collected in a nucleic acid amplification zone;
FIG. 9 is a typical fluorescence microscopy image of integrated nucleic acid extraction and ddPCR detection of Salmonella using the integrated flow channel digital microfluidic chip prepared in example 5, graphs A-D corresponding to different Salmonella sample volumes (5, 1, 0.5 and 0 μl), respectively.
Detailed Description
For further understanding of the present invention, the present invention will be described in detail with reference to the drawings and examples, but the present invention is not limited to these examples, and the present invention is not limited to the essential improvements and modifications made by those skilled in the art under the core teaching ideas of the present invention, and still falls within the scope of the present invention.
Fig. 1 is a schematic diagram of an integrated flow channel digital microfluidic chip disclosed by the invention, which consists of an upper polar plate 1 with a channel structure, a dielectric layer 2 and an electrode pattern layer 3, wherein the dielectric layer 2 is covered above the electrode pattern layer 3, and the upper polar plate 1 with the channel structure is positioned above the dielectric layer 2 and is sealed with the dielectric layer 2.
The upper polar plate 1 with the channel structure comprises a supporting layer 11, a middle conductive layer 12 and a channel structure layer 13 from top to bottom, and the upper polar plate 1 with the channel structure is also provided with a sample inlet 14 and a sample outlet 15.
The electrode pattern layer 3 includes a plurality of electrodes 31, and the plurality of electrodes 31 constitute an electrode pattern.
Fig. 2 is a schematic diagram of a processing flow of an upper plate with a channel structure layer in an integrated flow channel digital microfluidic chip disclosed in the present invention, wherein three processing methods are listed in the schematic diagram:
method (A):
(a) Placing a curable liquid material 16 over the mould 6;
(b) Connecting the support layer 11 with the intermediate conductive layer 12;
(c) Capping the intermediate conductive layer and the support layer prepared in step (b) onto the liquid material 16 in step (a) to form a thin film of the liquid material;
(d) The liquid material 16 is solidified to form a channel structure layer 13, and is connected with the supporting layer 11 and the conducting layer 12 to form an upper polar plate with a channel structure;
(e) And (3) turning down the whole upper polar plate with the channel structure in the step (d) from the die 6, and punching through holes at the positions of the sample inlet 14 and the sample outlet 15 by using a puncher.
In this method, the liquid material 16 may be formed into a thin film by spin coating or doctor blading, and then the intermediate conductive layer 12 and the supporting layer 11 may be capped.
Method (B):
(f) Placing a curable liquid material 16 over the mould 6;
(g) Forming the curable liquid material 16 into a thin film using spin coating or knife coating;
(h) Solidifying the liquid material film in the step (g) to form a channel structure layer 13;
(i) Connecting an intermediate conductive layer 12 on top of the channel structure layer 13 in step (h);
(j) Connecting a supporting layer 11 above the middle conductive layer 12 in the step (i) to form an upper polar plate with a channel structure;
(k) And (3) turning down the whole upper polar plate with the channel structure in the step (j) from the die 6, and punching through holes at the positions of the sample inlet 14 and the sample outlet 15 by using a puncher.
In this method, the middle conductive layer 12 may be connected to the supporting layer 11, and then connected to the channel structure layer 13 in the step (h), so as to form an upper plate with a channel structure.
Method (C):
(l) Placing a solid film 17 over the mould 6;
(m) processing a channel structure on the solid film 17 by a hot pressing method as the channel structure layer 13;
(n) connecting an intermediate conductive layer 12 on top of the channel structure layer 13 in step (m);
(o) further connecting a support layer 11 over the intermediate conductive layer of step (n) together to form an upper plate with a channel structure;
(p) turning down the whole upper polar plate with the channel structure in the step (o) from the die 6, and punching through holes at the positions of the sample inlet 14 and the sample outlet 15 by using a puncher.
In this method, the middle conductive layer 12 may be connected to the supporting layer 11, and then connected to the channel structure layer 13 in the step (m), so as to form an upper plate with a channel structure. For the solid-state thin film 17, the channel structure of the channel structure layer may also be processed by a laser etching or the like method commonly used in the art.
Example 1 Integrated flow channel digital microfluidic chip with polydimethylsiloxane as the channel structural layer material and photoresist as the dielectric layer material
a) And (3) processing a die: spin-coating photoresist (Microchem, SU-8 3050) on clean and dry monocrystalline silicon wafer, thickness of 100 μm, pre-drying, post-drying after exposure of mask (figure 3B), developing to remove the photoresist of unexposed part, and hard drying; before the die is used, trimethylchlorosilane (Allatin, C104813) is used for soaking, so that the die of the subsequent chip can be turned over conveniently.
b) Processing an upper polar plate with a channel structure: the mass ratio is 10:1 preparing a polydimethylsiloxane prepolymer and a curing agent (Michaelk, RTV 615), uniformly mixing, removing bubbles, taking 10g of the mixture and placing the mixture on a die processed in the step a), forming a thin layer with the thickness of about 120 mu m through spin coating, and heating and curing the thin layer to form a channel structure layer (H is 120 mu m, and H is 100 mu m); then spin-coating a conductive polymer PSS-PEDOT solution (Ossala, PH 1000) on the channel structure layer, and baking to form an intermediate conductive layer with the thickness of 100nm (the square resistance of the conductive surface is 150 ohms); pouring 30g of polydimethylsiloxane on the middle conductive layer, heating and curing to form a supporting layer, forming an upper polar plate with a channel structure together with the channel structure layer and the middle conductive layer, and turning down the whole body from the die; finally, punching a through hole at the position of the sample inlet and the sample outlet by using a puncher.
In this embodiment, the fluid channel 4, the micro-column array 5 and the chamber 6 are provided in the electrode pattern region and the non-electrode pattern region;
c) Electrode pattern layer processing: spin-coating positive photoresist (AZ 5214E, suzhou core Heng chip technology Co., ltd.) on clean and dry chromium plate (Yangshao light chromium plate Co., ltd., chromium layer thickness of 180 nm), and baking at 80deg.C for 2min; after exposure using the mask (FIG. 3A), baking at 80℃for 4min; developing, removing chromium and photoresist after maskless flooding exposure; finally, baking the mixture on a hot plate at 125 ℃ to remove the organic solvent.
d) Processing a dielectric layer: spin-coating a photoresist (Microchem, SU-8 1040) on the electrode pattern layer processed in step c) to a thickness of about 10 μm; then pre-baking, flood-exposing and post-baking are carried out; and finally, hardening in an oven to enable the dielectric layer and the electrode pattern layer to be closely adsorbed.
e) And (3) chip sealing: and b) performing plasma treatment on the upper polar plate with the channel structure in the step b) and the dielectric layer in the step d) (500V, 13.56MHz and 45 s), and performing alignment sealing to form the integrated flow channel digital micro-fluidic chip.
f) Hydrophobic modification: 1% of fluorine-containing silane coupling agent 1H, 2H-perfluoro decyl triethoxysilane (Allatin, P122385) is dissolved in fluorine oil (3M, FC-40) to prepare a hydrophobic modification solution; and e), introducing a hydrophobic modification solution into the integrated flow channel digital microfluidic chip in the step e), and finally drying the solvent to enable the channel structure layer and the medium layer to have hydrophobic surfaces.
Example 2 Integrated flow channel digital microfluidic chip with channel Structure layer Material of photo-curing Material (polyurethane acrylate) and Medium layer Material of photo-curing Medium layer Material (thiol-ene Polymer)
a) And (3) processing a die: exactly as in step a) of example 1.
b) Processing a supporting layer: a solution containing 59.4% urethane acrylate (chang materials, 6115J-80), 39.6% allyl methacrylate (sigma, 234931) and 1% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (sigma, 405655) was prepared by mass fraction; the solution was then placed between two clean glass plates under UV irradiation (365 nm,2.5 mW/cm) 2 ) Curing to form a photocurable thin layer having a thickness of about 50 μm; after removal of the upper glass plate, 30g of the glass plate was removed at a mass ratio of 5:1 and a curing agent (michaux, RTV 615) are poured over the photocurable film; and after heating and curing, the glass plate is tightly connected with the photo-curing thin layer, and the whole glass plate is taken off to form an elastic support body, and the elastic support body is cut into specified sizes as required to be used as a support layer for standby.
c) Processing an upper polar plate with a channel structure: preparing a light-cured liquid material containing 99% of polyurethane acrylic ester (Changxing materials Co., 6115J-80) and 1% of a photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-acetone (sigma, 405655) according to mass fraction, and placing the light-cured liquid material on a die processed in the step a); then, attaching an Indium Tin Oxide (ITO) conductive film (Roche gulo glass Co., ltd.) to one side of the photo-cured thin layer of the supporting layer in the step b) as an intermediate conductive layer (the ITO conductive material has a thickness of 200nm and the sheet resistance of the conductive surface is 7 ohms); then covering the photo-cured liquid material, lightly pressing to form a film with a thickness of about 120 μm, and irradiating with ultraviolet light (365 nm,2.5 mW/cm) 2 Solidifying for 200s to form a channel structure layer (H is 1) 20 μm, h is 100 μm), and forms an upper polar plate with a channel structure together with the supporting layer and the middle conductive layer, and the whole is turned down from the die; finally, punching a through hole at the position of the sample inlet and the sample outlet by using a puncher.
d) Electrode pattern layer processing: electrode pattern layers based on printed circuit boards (Printed Circuit Board, PCB) are custom processed in Shenzhen Jiedobang technology Co., ltd.
e) Processing a dielectric layer: preparing a thiol alkene solution containing 74% pentaerythritol tetra (3-mercaptopropionate) (aletin, P160529), 25% 1,3, 5-triallyl-1, 3, 5-triazine-2, 4,6 (1 h,3h,5 h) -trione (aletin, T123406) and 1% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (sigma, 405655) by mass fraction; spin-coating thiol-ene solution on the electrode pattern layer processed in step d), and finally under ultraviolet irradiation (365 nm,2.5 mW/cm) 2 200 s) is cured to form a dielectric layer having a thickness of about 5 μm.
f) And (3) chip sealing: aligning and tightly adhering the upper polar plate with the channel structure in the step c) and the medium layer in the step e), and irradiating with ultraviolet light (365 nm,2.5 mW/cm) 2 100 s) to form an integrated flow channel digital micro-fluidic chip.
g) Hydrophobic modification: 1% of hydrophobic light modification reagent 1H, 2H-perfluorodecanethiol (Allatin, P122296) is dissolved in fluorine oil (3M, FC-40) to prepare a hydrophobic modification solution; introducing a hydrophobic modification solution into the integrated flow channel digital microfluidic chip in the step f), and applying ultraviolet irradiation (365 nm,2.5 mW/cm) 2 100 s) followed by rinsing with fluorine oil and finally drying the solvent so that the channel structure layer and the dielectric layer have hydrophobic surfaces.
Example 3 Integrated flow channel digital microfluidic chip with thermoplastic polyurethane as the channel structural layer material and parylene as the dielectric layer material
a) And (3) processing a die: exactly as in step a) of example 1.
b) Processing an upper polar plate with a channel structure: processing a thermoplastic polyurethane material (basf, TPU Elastollan S65A) into a film having a thickness of about 120 μm by a hot-pressing method; placing the film on a die processed in the step a), and processing a channel structure on the film by using a hot press (150 ℃ C., 15 kPa) to form a channel structure layer (H is 120 mu m, H is 100 mu m); spin-coating a conductive polymer PSS-PEDOT solution (Ossala, PH 1000) on the channel structure layer, baking to form an intermediate conductive layer (thickness 100nm, sheet resistance of the conductive surface is 150 ohms); then a flat plate with the thickness of about 1mm is made of thermoplastic polyurethane material and is used as a supporting layer, and then the supporting layer is placed on the middle conducting layer and is connected with the channel structure layer and the middle conducting layer through a hot pressing method, so that an upper polar plate with a channel structure is formed together, and the whole upper polar plate is turned down from a die; finally, punching a through hole at the position of the sample inlet and the sample outlet by using a puncher.
c) Electrode pattern layer processing: exactly as in step c) of example 1.
d) Processing a dielectric layer: and C), evaporating a parylene C (Specialty Coating Systems, DPX-C) with a layer thickness of about 2 mu m on the electrode pattern layer processed in the step C) by a vacuum vapor deposition film plating machine (Specialty Coating Systems, PDS 2010) to serve as a medium layer.
e) And (3) chip sealing: and (3) aligning and tightly attaching the upper polar plate with the channel structure in the step b) and the medium layer in the step d), and forming the integrated flow channel digital micro-fluidic chip through thermocompression bonding.
f) Hydrophobic modification: and (3) according to mass fraction, dissolving 0.5% of Teflon (Kemu, AF 1600) in fluorine oil (3M, FC-40) to prepare a hydrophobic modification solution, introducing the hydrophobic modification solution into the integrated flow channel digital microfluidic chip in the step e), and finally drying the solvent to enable the channel structure layer and the medium layer to have hydrophobic surfaces.
Example 4 the channel Structure layer material was hydrophobic photo-curing material (perfluoropolyether acrylate), the Medium layer was an Integrated flow channel digital microfluidic chip with composite hydrophobic Medium layer
a) And (3) processing a die: exactly as in step a) of example 1.
b) Processing a supporting layer: exactly as in step b) of example 2.
c) Processing an upper polar plate with a channel structure: the composition contains 99% of perfluoropolyether acrylate (FLUOROLINK, MD 700) and 1% of light by mass fraction The initiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (sigma, 405655) is a hydrophobic photocurable liquid material and placed over the mold processed in step a); then spin-coating a PSS-PEDOT solution (Ossala, PH 1000) on the support layer of step b), baking to form an intermediate conductive layer (thickness of 100nm, sheet resistance of 150 ohms) and capping the intermediate conductive layer with the support layer, lightly pressing the intermediate conductive layer onto a photo-cured liquid material to form a film with a thickness of about 120 μm, and irradiating with ultraviolet light (365 nm,2.5 mW/cm) 2 130 s) to form a channel structure layer (H is 120 mu m, H is 100 mu m), and forming an upper polar plate with a channel structure together with the supporting layer and the middle conductive layer, wherein the whole body is turned down from the die; finally, punching a through hole at the position of the sample inlet and the sample outlet by using a puncher.
d) Electrode pattern layer processing: exactly as in step c) of example 1.
e) Processing a composite hydrophobic medium layer: preparing a thiol alkene mixture solution containing 74% pentaerythritol tetra (3-mercaptopropionate) (aletin, P160529), 25% 1,3, 5-triallyl-1, 3, 5-triazine-2, 4,6 (1 h,3h,5 h) -trione (aletin, T123406) and 1% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (sigma, 405655) by mass fraction; preparing a hydrophobic photo-curing agent containing 99.5% of perfluoropolyether acrylate (FLUOROLINK Co., MD 700) and 0.5% of photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (sigma, 405655); then spin-coating a thiol-ene mixture solution on the electrode pattern layer processed in step d), and irradiating with ultraviolet light (365 nm,2.5 mW/cm) 2 200 s) to form a thin layer having a thickness of about 5 μm; spin-coating hydrophobic photo-curing agent on the thin layer, and applying ultraviolet irradiation (365 nm,2.5 mW/cm) 2 400 s), the hydrophobic photo-curing agent is cured only on the surface of the thin layer of the close contact thiol-ene due to the strong oxygen inhibition, so as to form a thin hydrophobic layer with the thickness of about 2 mu m, and simultaneously, the thin hydrophobic layer is crosslinked with the thin thiol-ene; and finally, washing the uncured photo-curing agent by using absolute ethyl alcohol, flushing with deionized water, drying by nitrogen, and baking at 80 ℃ for 15min to form the composite hydrophobic medium layer.
f) And (3) chip sealing: carrying out step c) withThe upper polar plate of the channel structure is aligned and clung to the composite hydrophobic medium layer in the step e), and ultraviolet irradiation (365 nm,2.5 mW/cm) is applied in a nitrogen environment 2 100 s) to form the integrated flow channel digital micro-fluidic chip.
In this embodiment, since the channel structure layer is made of a hydrophobic photo-curing material, and the dielectric layer is a composite hydrophobic dielectric layer, the channel structure layer has a hydrophobic surface, so that the channel of the integrated flow channel digital microfluidic chip can be ensured to have good hydrophobic characteristics without hydrophobic modification.
Example 5 Integrated flow channel digital microfluidic chip with channel structural layer made of hydrophobic light-cured material (perfluoropolyether acrylate) and dielectric layer made of composite hydrophobic dielectric layer
a) And (3) processing a die: spin-coating photoresist (Microchem, SU-83050) on the clean and dry silicon wafer to a thickness of about 100 μm, and then performing pre-baking; attaching the first layer of mask (figure 6A) to the photoresist, exposing for 13s, and post-baking; spin-coating photoresist (Microchem, SU-8 3050) again to a thickness of about 100 μm, and pre-baking; placing the second layer mask (fig. 6B) on a single-sided photoetching machine, aligning the first layer photoresist pattern with the second layer mask by using an alignment system, exposing for 13s, and post-baking; finally developing and hard drying; before the die is used, trimethylchlorosilane (Allatin, C104813) is used for soaking, so that the die of the subsequent chip can be turned over conveniently.
b) Processing a supporting layer: exactly as in step b) of example 2.
c) Processing an upper polar plate with a channel structure: preparing a hydrophobic light-cured material solution containing 99% of perfluoropolyether acrylate (FLUOROLINK Co., MD 700) and 1% of photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (sigma, 405655) by mass fraction; dripping the mixture on the die processed in the step a); then, attaching an ITO conductive film (Roche gulo glass Co., ltd.) with the same size as the electrode pattern area on one side of the photo-curing thin layer of the support layer prepared in the step b) as an intermediate conductive layer (the thickness of the ITO conductive material is 200nm, and the square resistance of the conductive surface is 7 ohms); then covering the liquid material on the solution of the hydrophobic light-cured material, and lightly pressing the liquid material to form a film with the thickness of about 220 mu m; under ultraviolet irradiation (3) 65nm,2.5mW/cm 2 ) Solidifying the hydrophobic light solidifying material solution to form a channel structure layer (H is 220 mu m, H is 200 mu m), forming an upper polar plate with a channel structure together with the supporting layer and the middle conducting layer, and turning down the whole body from the die; finally, punching a through hole at the position of the sample inlet and the sample outlet by using a puncher.
d) Electrode pattern layer processing: the electrode pattern layer was processed using the mask shown in fig. 7, and the processing procedure was exactly the same as in step c) of example 1.
e) Processing a composite hydrophobic medium layer: exactly as in step e) of example 4.
f) And (3) chip sealing: exactly as in step f) of example 4.
In this embodiment, since the channel structure layer is made of a hydrophobic photo-curing material, and the dielectric layer is a composite hydrophobic dielectric layer, the channel structure layer has a hydrophobic surface, so that the channel of the integrated flow channel digital microfluidic chip can be ensured to have good hydrophobic characteristics without hydrophobic modification.
Example 6 channel Structure layer Material is hydrophobic Photocurable Material (perfluoropolyether acrylate), medium layer is Integrated flow channel digital microfluidic chip of composite hydrophobic Medium layer
The preparation method of this example is substantially the same as that of example 5, except that the thickness of the film of the photocurable liquid material in step c) is about 250. Mu.m, and the channel structure layer is formed with H of 250. Mu.m, and the corresponding (H-H) of 50. Mu.m.
Under this parameter, the droplet can be manipulated to move, generate, split, and fuse, but the droplet movement speed is slower than in example 5, probably because of the larger impedance between the droplet and the conductive layer caused by the channel structure layer material, resulting in poor driving performance of the droplet.
Example 7 Integrated flow channel digital microfluidic chip with channel structure layer made of hydrophobic light-cured material (perfluoropolyether acrylate) and dielectric layer made of composite hydrophobic dielectric layer
The preparation method of this example is substantially the same as that of example 5, except that the thickness of the photo-curable liquid material film in step c) is about 280. Mu.m, the channel structure layer is formed with H of 280. Mu.m, and the corresponding (H-H) of 80. Mu.m. With this parameter, the droplet movement speed is slow, and it is difficult to achieve the droplet splitting, because the impedance between the droplet and the conductive layer caused by the channel structure layer material may be excessively large, resulting in deterioration of the driving performance of the droplet, even loss of the droplet splitting function.
It can be found from examples 5 to 7 that, (H-H) needs to be within a proper range in the channel structure layer of the integrated flow channel digital microfluidic chip to satisfy good droplet driving performance, and as (H-H) increases, the droplet movement effect is worse.
Example 8 Integrated flow channel digital microfluidic chip with channel structural layer made of hydrophobic light-cured material (perfluoropolyether acrylate) and dielectric layer made of composite hydrophobic dielectric layer
The preparation method of this example is substantially the same as that of example 4, except that the conductive polymer PSS-PEDOT solution (Ossala, PH 1000) spin-coated on the support layer in step c) has a thickness of about 300nm, and an intermediate conductive layer is formed after baking, and the sheet resistance is measured to be about 15 ohms. Although the conductive material is thicker and has poorer transparency under the parameter, the movement, generation, splitting and fusion of the liquid drops can be better carried out, so the conductive material can be adopted in the application occasions with low requirements on the transparency of the chip and no optical detection.
Example 9 Integrated flow channel digital microfluidic chip with channel structural layer made of hydrophobic light-cured material (perfluoropolyether acrylate) and dielectric layer made of composite hydrophobic dielectric layer
The preparation method of this example is substantially the same as that of example 4, except that the conductive polymer PSS-PEDOT solution (Ossala, PH 1000) spin-coated on the support layer in step c) has a thickness of about 50nm, and an intermediate conductive layer is formed after baking, and the sheet resistance is measured to be about 500 ohms. Under this parameter, movement, generation, splitting, and fusion of the droplets can be achieved, but the droplet movement speed is slow, probably due to the small thickness of the conductive material, the poor conductivity of the formed thin layer, and the large impedance.
According to embodiments 4, 8 and 9, it can be found that the thickness of the conductive material and the sheet resistance of the conductive layer in the integrated flow channel digital microfluidic chip need to be within a proper range to meet the good droplet driving performance, and the smaller the sheet resistance, the better the droplet movement effect.
Application example 1 continuous fluid sample injection and sample discharge in integrated flow channel digital micro-fluidic chip
The integrated flow channel digital microfluidic chip prepared in the embodiment 1 is arranged on a digital microfluidic droplet controller, and the voltage, frequency and time of an electrode can be controlled through software MicroFluid matched with the digital microfluidic droplet controller, so that droplet movement is controlled; connecting an injector on the injection pump with a sample inlet and a sample outlet of the chip by using a polytetrafluoroethylene tube, and controlling sample injection and sample discharge of continuous fluid in the chip by using the injection pump;
fluorine oil (3M, FC-40) is introduced into and filled in the integrated flow channel digital micro-fluidic chip in advance, and air is discharged; pumping 10 mu L of aqueous solution into the chip at a flow rate of 500 mu L/h, and entering an electrode pattern area from a sample injection channel; then controlling the electrode voltage to form liquid drops, moving the liquid drops to a sample outlet channel port, and pumping fluorine oil from a sample inlet at a flow rate of 500 mu L/h so as to push the liquid drops to enter the sample outlet channel from an electrode area, thereby realizing continuous sample outlet of fluid;
FIG. 4 is a photomicrograph of continuous fluid control in an integrated fluidic channel digital microfluidic chip prepared in example 1, wherein (a) aqueous solution enters the electrode pattern region from the channel and (b) aqueous solution enters the channel from the electrode pattern region; experiments show that the integrated flow channel digital micro-fluidic chip provided by the invention realizes that a channel structure and an electrode pattern are integrated on a totally-enclosed chip, and is easy to combine with an external interface, so that continuous sample inlet and outlet fluid control is facilitated, and the integrated flow channel digital micro-fluidic chip has the capability of flow channel-digital bidirectional liquid drop transportation.
Application example 2 droplet driving in integrated flow channel digital microfluidic chip
The integrated flow channel digital microfluidic chip prepared in the embodiment 4 is arranged on a digital microfluidic droplet controller, a conducting layer of the integrated flow channel digital microfluidic chip is connected with a grounding electrode of the digital microfluidic droplet controller, working voltage is set to be 150V, frequency is set to be 10MHz on software MicroFluid matched with the digital microfluidic droplet controller, and the opening and closing of related electrodes are controlled, so that droplets in the integrated flow channel digital microfluidic chip can be driven to split, fuse, move and the like;
fluorine oil (3M, FC-40) is introduced into and filled in the integrated flow channel digital micro-fluidic chip in advance, and air is discharged; pumping 15 mu L of aqueous solution into the chip to form a large droplet crossing three electrodes, closing the middle electrode, opening the two electrodes, and splitting into two smaller droplets; then the middle electrode is opened, the two side electrodes are closed, and the two small liquid drops are fused into one large liquid drop; with the sequential opening of adjacent electrodes, the droplets can rapidly move to the opened electrodes;
Fig. 5 is a photomicrograph of the driving of droplets in an integrated fluidic digital chip prepared in example 4, where (a) is a droplet splitting process, (b) is a droplet fusion process, and (c) is a droplet moving process. The droplet generation process is similar to the droplet break up process. Experiments show that the integrated flow channel digital micro-fluidic chip forms a bipolar plate digital micro-fluidic structure by introducing the grounding conductive layer, and can realize all basic functions of droplet generation, splitting, movement and fusion.
Application example 3 salmonella nucleic acid extraction and ddPCR detection
An integrated flow channel digital microfluidic chip was prepared using example 5, and integrated nucleic acid extraction and droplet digital PCR (ddPCR) detection of salmonella was performed. The channel structure of the chip is shown in fig. 6, and comprises a digital microfluidic nucleic acid extraction region 1 and a digital nucleic acid detection region 2. The digital microfluidic nucleic acid extraction region 1 is provided with four sample inlets 11, a nucleic acid extraction chamber 12 and a nucleic acid solution sample outlet channel 13; the digitized nucleic acid detection zone 2 includes a sample distribution channel 21, an oil phase channel 22, a nucleic acid amplification zone 23, and a sample outlet 24. The electrode pattern of the electrode pattern layer is located below the nucleic acid extraction chamber as shown in FIG. 7. The specific procedures of nucleic acid extraction and ddPCR detection are as follows:
The integrated flow channel digital microfluidic chip prepared in the embodiment 5 is arranged on a digital microfluidic droplet controller, and the voltage, frequency and time of an electrode can be controlled through software MicroFluid matched with the digital microfluidic droplet controller, so that droplet movement is controlled; connecting an injector on the injection pump with a sample inlet and a sample outlet of the chip by using a polytetrafluoroethylene tube, and controlling the sample injection, the sample outlet and the preparation of microdroplets of continuous fluid in the chip by using the injection pump; in order to perform a nucleic acid extraction experiment by a magnetic bead method, an electromagnet is arranged below a chip, and is powered by 24V direct current voltage for fixing magnetic beads. Before the experiment, fluorine oil is introduced into and fills the chip in advance, and air is discharged.
Adding salmonella culture solution into the tissue digestion solution and the lysis solution, and uniformly mixing to obtain a sample solution; preparing a PCR reaction solution comprising 2 mu L of nuclease-free water, 2.5 mu L of 2 Xnucleic acid amplification reaction solution and 0.5 mu L of 10 Xprimer probe reaction solution; at the four sample inlets, 2.5. Mu.L of magnetic bead solution, 10. Mu.L of buffer solution, 10. Mu.L of rinse solution, 5. Mu.L of PCR reaction solution (here the PCR reaction solution is also used as eluent to simplify the operation and facilitate the subsequent PCR reaction) are loaded respectively; and 5 mu L of sample solution is injected into the chip and mixed with the magnetic bead solution, and then the nucleic acid extraction processes of sample pyrolysis, nucleic acid adsorption, buffer solution cleaning, rinsing liquid rinsing and nucleic acid elution are sequentially carried out in a digital microfluidic nucleic acid extraction area.
After nucleic acid extraction is completed, transporting liquid drops containing nucleic acid and PCR reaction liquid to a nucleic acid solution sample outlet area of a digital microfluidic nucleic acid extraction area, continuously introducing an oil phase from a sample inlet opposite to the nucleic acid solution sample outlet channel to push the liquid drops to enter a sample distribution channel of a digital nucleic acid detection area, and then generating water-in-oil droplets at a T-shaped droplet generation structure with the oil phase solution (HFE 7500 containing 1% of surfactant Pico-Surf, 100 mu L/h); transferring the chip onto a hot plate for PCR reaction, and setting a temperature control program as follows: maintaining at 95deg.C for 5min: then carrying out heat circulation for 40 times at 95 ℃ for 15s and 60 ℃ for 30s, and finally cooling to 10 ℃; after the PCR reaction is completed, the chip is placed on an inverted fluorescence microscope and a fluorescent image of the droplet array in the chamber is acquired.
Fig. 8 is a photomicrograph of droplet generation within an integrated fluidic channel digital microfluidic chip. The nucleic acid solution extracted in the digital microfluidic nucleic acid extraction zone can directly enter a sample distribution channel of the digital nucleic acid detection zone through a sample outlet channel, and stably generates water-in-oil droplets at a T-shaped droplet generation structure together with the oil phase pumped from the oil phase channel (figure 8A), and then is collected in a nucleic acid amplification zone (figure 8B) for ddPCR experiments and fluorescence detection.
FIG. 9 is a typical fluorescence image of Salmonella after integrated nucleic acid extraction and ddPCR experiments in an integrated flow channel digital microfluidic chip. In the figure, A to D correspond to different salmonella sample amounts (5. Mu.L, 1. Mu.L, 0.5. Mu.L, 0. Mu.L). The results show that as the sample volume increases, the concentration of extracted DNA increases, and therefore the number of fluorescent droplets after ddPCR increases. The results fully demonstrate the effectiveness of salmonella nucleic acid extraction and detection.
The results show that the integrated flow channel digital microfluidic chip can realize the organic integration of flow channel microfluidic and digital microfluidic, has the capability of controlling continuous fluid and discrete liquid drops, and can conveniently integrate various functional units, so that the integrated flow channel digital microfluidic chip has wider application prospects in the fields of digital microfluidic, liquid drop microfluidic, biochemical reaction, biomedical research and the like.
The foregoing is a preferred embodiment of the present invention and is not to be construed as limiting thereof. It will be apparent to those skilled in the art that numerous modifications and variations can be made without departing from the principles of the invention, and such modifications and variations are considered to be within the scope of the invention.

Claims (8)

1. The utility model provides an integrated runner digital micro-fluidic chip, includes electrode pattern layer and covers in dielectric layer on the electrode pattern layer, its characterized in that:
the upper polar plate is sealed with the dielectric layer and provided with a channel structure;
the upper polar plate with the channel structure comprises a supporting layer, a middle conductive layer and a channel structure layer from top to bottom, and the middle conductive layer can be connected with an external electrode;
the channel structure of the channel structure layer is positioned above the electrode pattern of the electrode pattern layer or selectively added above the non-electrode pattern area of the electrode pattern layer;
when a channel structure of the channel structure layer is positioned above an electrode pattern of the electrode pattern layer and digital microfluidic droplet control is involved, the thickness of the channel structure layer is set to be H, and the height of the channel structure is set to be H, wherein H is selected from 20-500 mu m, and 0 < (H-H) is less than or equal to 50 mu m;
the thickness of the conductive material in the middle conductive layer is 100-200 nm, and the sheet resistance of the formed conductive surface is 5-150 ohms.
2. The integrated fluidic digital chip of claim 1 wherein:
the material of the channel structure layer is one or more selected from polydimethylsiloxane, a photo-curing material and a thermoplastic material.
3. The integrated fluidic digital chip of claim 2 wherein:
the channel structure layer has a hydrophobic surface;
when the material of the channel structure layer is a non-hydrophobic material, the surface treatment of the channel structure layer can be performed by a hydrophobic modification reagent.
4. The integrated fluidic digital chip of claim 1 wherein:
the middle conductive layer is selected from a film with a conductive material coated on the surface or a thin layer formed by processing the conductive material;
the conductive material is selected from one or more of conductive oxide, conductive metal, conductive polymer and graphene.
5. The integrated fluidic digital chip of claim 1 wherein:
the material of the supporting layer is one or more selected from polydimethylsiloxane, an elastic supporting body, plastics and glass;
and the upper polar plate with the channel structure is also provided with a sample inlet and a sample outlet.
6. The integrated fluidic digital chip of claim 1 wherein:
the electrode pattern layer is an electrode pattern formed by a plurality of electrodes processed on the substrate, and is connected with an external control circuit;
The substrate is selected from glass, silicon wafer, printed circuit board, plastic board or rubber board;
the materials of the plurality of electrodes are selected from one or more of conductive oxides, conductive metals, conductive polymers and graphene;
the dielectric layer is a hydrophobic dielectric layer material thin layer or a composite hydrophobic dielectric layer prepared by using a photo-curing dielectric layer material;
the relative dielectric constant of the dielectric layer material is selected from 3-10;
the relative dielectric constant of the photo-curing dielectric layer material is selected from 3-10;
the thickness of the dielectric layer is selected from 1-50 mu m.
7. A method for manufacturing an integrated fluidic channel digital microfluidic chip according to any one of claims 1 to 6, comprising: processing an upper polar plate with a channel structure, processing a dielectric layer, processing an electrode pattern layer and sealing a chip, wherein the upper polar plate with the channel structure is processed according to one of the following methods:
a) Placing a curable liquid material on a die, forming a film by a spin coating or knife coating method, then covering the middle conductive layer and the supporting layer, or directly covering the middle conductive layer and the supporting layer on the liquid material to form a film, and curing to form an upper polar plate with a channel structure;
B) Placing a curable liquid material on a die, forming a film by a spin coating or knife coating method, curing to form a channel structure layer, and then connecting the middle conductive layer and the supporting layer to form an upper polar plate with a channel structure;
c) And processing a channel structure on the solid film by a hot-pressing method or a laser etching method to form a channel structure layer, and connecting the channel structure layer with the middle conductive layer and the supporting layer to form an upper polar plate with the channel structure.
8. An application of the integrated flow channel digital microfluidic chip according to any one of claims 1-6 in the fields of digital microfluidic, droplet microfluidic, biochemical reaction and biomedical research.
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CN110653011A (en) * 2019-09-30 2020-01-07 浙江大学 Reusable double-layer digital microfluidic chip based on hydrophobic film and rapid preparation method
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