Photocured digital detection chip and application thereof

Abstract

The application discloses a kind of photo-curing digital detection chip and its application, the chip includes chip main body and sealing substrate, chip main body includes support layer and channel structure layer;Channel structure layer is the thin layer prepared by photo-curing material after photo-curing, and channel structure is processed on the thin layer;Photo-curing material includes raw material containing photo-curing functional group and photo-initiator;Photo-curing material has air permeability after photo-curing;Photo-curing functional group is selected from one or more of mercapto functional group, alkenyl functional group, acrylate functional group, methacrylate functional group, vinyl ether functional group, epoxy functional group.The chip solves the problem of sample injection difficulty of plastic chamber type digital detection chip, and can be injected by positive pressure, negative pressure and the combination of positive and negative pressure, and the injection mode is flexible and simple;Meanwhile, chip processing process is simple, fast, high throughput and low cost, which makes up for the deficiency of high cost and difficult to quickly prepare PDMS chip;The photo-curing digital detection chip can be widely applied to nucleic acid, protein and other biomolecule detection field, and cell, microorganism culture field.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chip, and particularly relates to a light-cured digital detection chip and application thereof. BACKGROUND

[0002] Digital PCR (dPCR) is known as the third generation technology. It is to uniformly distribute the sample to be detected into many independent small chambers, so that each small chamber contains at most one target nucleic acid molecule, after PCR amplification, only the small chamber containing the target nucleic acid molecule emits fluorescence, therefore, as long as the number of these fluorescent small chambers is counted, the copy number of the target nucleic acid molecule can be obtained. dPCR can realize the absolute quantification of nucleic acid molecules, has the outstanding advantages of sensitivity, accuracy, etc., and has very good application prospect in many aspects such as clinical diagnosis, biomedical research, microbial detection, food safety detection, etc.

[0003] In addition to dPCR, many digital detection methods based on isothermal amplification technology have emerged recently, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), crossing priming amplification (CPA), rolling circle amplification (RCA), strand displacement amplification (SDA), and CRISPR-based nucleic acid detection methods, etc. In addition, there are also digital enzyme-linked immunoassay (Digital ELISA) and other protein molecule detection technologies.

[0004] At present, the mainstream digital detection chip can be mainly divided into droplet type and chamber type according to the sample distribution mode. Among them, the chamber type digital detection chip is to distribute the sample into independent micro-wells or micro-chambers, which has the advantages of uniform chamber size, stable separation, convenient real-time fluorescence observation, etc. However, the current chamber type digital detection chip still mainly uses single crystal silicon wafer and thermoplastic plastic material. The former has complex processing technology and high cost, while the latter has low cost, but due to the poor air permeability of thermoplastic plastic itself, it is difficult to distribute the sample into the micro-chamber. Polydimethylsiloxane (PDMS) has good air permeability, and the chip made of it can well solve the sample distribution problem. However, the heat curing speed of this material is slow, the chip is difficult to mass-produce, and the cost is also high, which seriously hinders its commercialization and popularization.

[0005] Therefore, it is still urgent to develop a digital detection chip with good air permeability, rapid molding and low cost. SUMMARY

[0006] In view of the above problems, the application discloses a photocured digital detection chip. Through optimization of the photocured material and the chip design, the chip has good air permeability, so that sample distribution in digital detection is more convenient and easy. In addition, the preparation process of the chip is simple, fast and low in cost.

[0007] The specific technical solutions are as follows:

[0008] A photocured digital detection chip comprises a chip main body and a sealing base.

[0009] The chip main body comprises a support layer and a channel structure layer.

[0010] The channel structure layer is a thin layer prepared by photocuring of a photocured material, and the thin layer is processed with a channel structure.

[0011] The photocured material comprises a raw material containing a photocured functional group and a photoinitiator.

[0012] The photocured material has air permeability after photocuring.

[0013] The photocured functional group is selected from one or more of a mercapto functional group, an alkenyl functional group, an acrylate functional group, a methacrylate functional group, a vinyl ether functional group and an epoxy functional group.

[0014] In the application, the thin layer prepared by photocuring has a reticular structure, and the reticular structure is a polymer crosslinked body with many mesh structures, and the mesh is selected from mesopores and micropores with a pore size of not more than 20 nm.

[0015] The thin layer prepared by the photocured material mainly permeates oxygen and nitrogen; the oxygen permeability of the thin layer prepared by the photocured material is selected from 5*10 5 ~ 2*10 9 cm 3 · μm / mm 2 · 24h*0.1MPa; and the nitrogen permeability is selected from 1*10 5 ~ 2*10 9 cm 3 · μm / mm 2 · 24h*0.1MPa.

[0016] The raw material containing the photocured functional group is a monomer and / or an oligomer.

[0017] Preferably, one or more of tetra(3-mercaptopropionic acid) pentaerythritol ester, tris[2-(3-mercaptopropionyloxy)ethyl] isocyanurate, trimethylolpropane tri(3-mercaptopropionate), triallyl isocyanurate, trimethylolpropane diallyl ether, perfluoro polyether acrylate, polysiloxane acrylate, polysiloxane methacrylate, mercapto-containing polysiloxane, alkenyl-containing polysiloxane is used.

[0018] Further preferably, the raw material containing photo-curable functional groups is selected from mercapto-containing polysiloxane and alkenyl-containing polysiloxane. In the photo-curing process, the mercapto group and the alkenyl group cross-link to form a space network structure, so that the material has good air permeability; the number of mercapto and alkenyl functional groups of the mercapto-containing polysiloxane and the alkenyl-containing polysiloxane is greater than or equal to 2; and the molar ratio of the mercapto and alkenyl functional groups in the mixture of the mercapto-containing polysiloxane and the alkenyl-containing polysiloxane is selected from 1:(0.5-2.0).

[0019] The photoinitiator is selected from one or more of the conventional types in the art, such as benzoin compounds, alkylbenzophenone compounds, acyl phosphine oxides.

[0020] Preferably, the mass fraction of the photoinitiator is 0.5-2.0% based on the total mass of the photo-curable material.

[0021] Preferably, the photo-curable material further comprises a leveling agent, which is specifically selected from one or more of alkyl-modified silicone monomers, ether bond-modified silicone monomers, ester group-modified silicone monomers, polydimethylsiloxane, polyacrylate, polyether siloxane copolymer, polyester siloxane copolymer. It is found through experiments that the addition of the leveling agent can increase the mold turning precision and success rate.

[0022] Preferably, the mass fraction of the leveling agent is selected from 0.1%-2.0% based on the total mass of the photo-curable material.

[0023] Further preferably, the leveling agent is selected from 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane.

[0024] The photo-curable material can also be selectively added with a toughening agent, so that the photo-cured material has stronger toughness, enhances the stability of the chip structure, and is convenient for chip demolding;

[0025] The toughening agent is selected from one or more of the conventional types in the art, such as maleic acid diallyl ester, bisphenol A ethoxy acid diacrylate, isobornyl acrylate, methyl isobornyl acrylate, trimethylolpropane triacrylate, tricyclo[5.2.1.02 2,6 ]decane dimethylol acrylate, 1,6-hexanediol diacrylate, 1,10-decanediol diacrylate, polyurethane acrylate.

[0026] Preferably, the mass fraction of the toughening agent is selected from 0.1% to 30% based on the total mass of the photocuring material.

[0027] In the present application:

[0028] The channel structure layer comprises fluid channels for fluid flow and a microcavity array for accommodating samples;

[0029] The fluid channels comprise sample distribution channels and anti-evaporation channels;

[0030] The sample distribution channels comprise a tree-shaped channel, a plurality of branch channels, a sample inlet and a sample outlet; the tree-shaped channel is in communication with each branch channel at the end thereof, and the branch channels extend into the microcavity array region and are connected to the microcavities in the microcavity array.

[0031] Preferably, the tree-shaped channel adopts a binary tree structure, which can ensure the flow resistance balance of each branch of the channel and is conducive to the sample injection process of the chip.

[0032] The anti-evaporation channels surround the sample distribution channel region and the microcavity array region, and the height thereof is greater than or equal to the cavity height h2 of the microcavity array.

[0033] Preferably:

[0034] The distance h1 between the top end of the microcavity array and the top end of the channel structure layer is selected from 20 to 300 μm. If h1 is too small, the light-curing material with certain air permeability in the channel structure layer is less, and the ability to maintain negative pressure after vacuum treatment is reduced; if h1 is too large, the anti-evaporation layer is too far away from the microcavity array, and the anti-evaporation performance of the chip will be weakened.

[0035] The cavity height h2 of the microcavity array region is selected from 20 to 200 μm; h2 is 1 to 3 times the height h3 of the branch channel. Within this height range, the chip works better. If the ratio is too small, the liquid in the microcavity will flow back into the sample distribution channel, and the cavities may be interconnected, resulting in unreliable digital detection results; if the ratio is too large, the sample injection rate of the chip will be affected, and the sample injection time will be prolonged.

[0036] The microcavity array is selected from a microcavity or a micro-well microstructure array.

[0037] Some digital detection methods involve high temperature conditions, at which the aqueous solution in the microcavity is prone to evaporation, resulting in loss of the solution in the microcavity.

[0038] Preferably:

[0039] The support layer and the channel structure layer are further provided with an anti-evaporation layer, which is located above the channel structure area of the channel structure layer and has an area greater than or equal to that of the channel structure area.

[0040] Preferably, the anti-evaporation layer only needs to be located above the microcavity array area of the channel structure layer and has an area greater than or equal to that of the microcavity array area of the channel structure layer, and can completely cover the microcavity array area.

[0041] The anti-evaporation layer is a gas-impermeable film, such as a PP film, etc., and is fixedly connected with the support layer and the channel structure layer, respectively.

[0042] The surface of the gas-impermeable film is further provided with an adhesive material selected from one or more of silicone materials, acrylic materials, polyester materials, and rubber materials; preferably, the adhesive material is a silicone material.

[0043] Preferably:

[0044] The anti-evaporation layer has good transparency, and the light transmittance is not less than 90%, so as to facilitate observation and optical detection.

[0045] The thickness h4 of the anti-evaporation layer is selected from 10 to 150 μm.

[0046] In the present application:

[0047] The material of the support layer is selected from one or more of glass, plastic, rubber, and photocurable material.

[0048] The sealing base can be a single base layer or a composite layer composed of a sealing layer and a base layer, and the material is selected from one or more of glass, plastic, rubber, polydimethylsiloxane, double-sided adhesive tape, and photocurable material.

[0049] When the sealing layer is introduced, the sealing layer is made of photocurable material or double-sided adhesive tape.

[0050] When the sealing layer is made of photocurable material, the sealing base is processed by one of the following methods:

[0051] a) The liquid photocurable material is spread on the base layer to form a thin layer, and is given light irradiation to be cured to form the sealing base;

[0052] b) The photocurable material film is pasted on the base layer to form the sealing base.

[0053] When the channel structure layer is made of material with a polysiloxane skeleton, glass or PDMS can also be used as the sealing base and is bonded to the chip body through plasma treatment.

[0054] The chip body is processed by one of the following methods:

[0055] A) placing the configured liquid photocuring material on the mold, using the method of doctor blading or spin coating to spread the liquid photocuring material into a thin layer, then covering the support layer and the evaporation prevention layer onto the thin layer, or directly covering the support layer and the evaporation prevention layer onto the liquid photocuring material, and applying pressure to the support layer to make the liquid photocuring material form a thin layer, then curing the liquid photocuring material to form a channel structure layer, and together with the support layer and the evaporation prevention layer to form a chip main body;

[0056] B) placing the configured liquid photocuring material on the mold, using the method of doctor blading or spin coating to spread the liquid photocuring material into a thin layer, curing the liquid photocuring material to form a channel structure layer, then connecting the evaporation prevention layer and the support layer to form a chip main body;

[0057] After the sealing of the base and the chip main body is stable, the photocured digital detection chip is prepared.

[0058] The sealing method includes common methods such as plasma bonding, donor bonding, light sealing, heat sealing, adhesion, etc.

[0059] The application further discloses a detection method of the photocured digital detection chip, which comprises the following steps:

[0060] (1) using negative pressure and / or external pressure to drive the digital detection reaction solution to be quickly distributed into the microcavity array in the channel structure, then injecting separation oil and replacing the liquid in the sample distribution channel in the channel structure after the sample injection is completed, and after the fluid is stable, photocuring is performed until the separation oil is cured, and the sample injection process is completed;

[0061] The separation oil is selected from one or more of the following: tetra (3-mercaptopropionic acid) pentaerythritol ester, triallyl isocyanurate, maleic acid diallyl ester, bisphenol A ethoxy acid dipropylene acid, isobornyl acrylate, methyl isobornyl acrylate, trimethylolpropane triacrylate, tricyclo [5.2.1.0 2,6 ] decane dimethylol acrylate, neopentyl glycol polymethyl oxirane diacrylate, 1,6-hexanediol diacrylate, polyurethane acrylate, mercapto-containing polysiloxane, alkenyl-containing polysiloxane, acrylate polysiloxane, and methacrylate polysiloxane.

[0062] The viscosity of the separation oil is 0.5-100 cSt.

[0063] (2) placing the digital detection chip on a hot plate, setting the temperature rising and falling program of the hot plate according to the digital detection reaction to be taken, and performing the digital detection reaction; after the digital detection reaction is completed, placing the chip under a fluorescence field, and collecting the fluorescence image of the microcavity array of the chip.

[0064] The sample injection process of step (1) is to fill the sample distribution channel and the microcavity array with the digital detection reaction solution, and then to flush the sample solution in the sample distribution channel with the separation oil and fill the whole sample distribution channel, so that the digital detection reaction solution only exists in the microcavity array.

[0065] The negative pressure injection is to place the photocured digital detection chip (with the inlet and outlet sealed) in a vacuum device to remove the gas inside the chip, and then to open the inlet under normal pressure and use the atmospheric pressure to load the digital detection reaction solution and the separation oil into the chip;

[0066] The positive pressure injection is to seal the outlet of the photocured digital detection chip, and to load the digital detection reaction solution and the separation oil into the chip by the external applied pressure and the air permeability of the chip itself;

[0067] The positive and negative pressure combined injection is to place the photocured digital detection chip (with the inlet and outlet sealed) in a vacuum device to remove the gas inside the chip, and then to open the inlet under normal pressure and load the digital detection reaction solution and the separation oil into the chip by the external applied pressure and the air permeability of the chip itself;

[0068] Preferably,

[0069] The sample injection method of the photocured digital detection chip is the positive and negative pressure combined injection, which has a faster injection rate and a stable injection effect.

[0070] The separation oil is an oil phase that is immiscible with the digital detection reaction solution, and is selected from one or more of mineral oil, silicone oil, fluorine oil and photocured oil;

[0071] The separation oil also needs to add a photoinitiator, and the specific type is selected from the conventional types in the art. The mass fraction of the photoinitiator in the total mass of the separation oil is 0.5-2.0%.

[0072] Preferably, the separation oil includes one or more of tetra (3-mercaptopropionic acid) pentaerythritol ester, triallyl isocyanurate, maleic acid diallyl ester, bisphenol A ethoxy acid dipropargyl acid, isobornyl acrylate, methyl isobornyl acrylate, trimethylolpropane triacrylate, tricyclo [5.2.1.0 2,6 ] decane dimethylol acrylate, neopentyl glycol polymethyl oxirane diacrylate, 1,6-hexanediol diacrylate, polyurethane acrylate, mercapto-containing polysiloxane, alkenyl-containing polysiloxane, acrylate polysiloxane and methacrylate polysiloxane.

[0073] Further preferably, tetra (3-mercaptopropionic acid) pentaerythritol ester and maleic acid diallyl ester are included, and the molar ratio of the mercapto and alkenyl functional groups is selected from 1:(0.5-2.0).

[0074] The light-cured digital detection chip can be widely applied to the field of biomolecule detection such as nucleic acid and protein, and the field of cell and microbial culture. When applied to the field of cell and microbial culture, the size of the microchamber in the microchamber array can be adjusted according to requirements, and the anti-evaporation channel, the anti-evaporation layer and the separation oil can be selected according to actual requirements.

[0075] Compared with the prior art, the present application has the following beneficial effects:

[0076] The present application discloses a light-cured digital detection chip. The chip is formed by irradiating the chip body with ultraviolet light using a preferred light-cured material. The chip body material has certain air permeability, which makes it more convenient and easy to distribute the sample into the microchamber array. The sample can be introduced by positive pressure, negative pressure or a combination of positive and negative pressure. The sample introduction method is flexible and simple, which effectively solves the problem of difficult sample distribution of the plastic chamber type digital detection chip. Taking the combination of positive and negative pressure as an example, after simple negative pressure pretreatment of the chip, the digital detection reaction solution can be quickly distributed into the microchamber array under the driving of positive pressure, which greatly reduces the sample distribution time. The light-cured digital detection chip can also use light-cured oil phase as separation oil, which can be stably connected with the chip surface after curing, so that the separation effect is better, the cross talk between the chambers can be reduced, and the accuracy of the detection result is ensured.

[0077] By adding a specific type of leveling agent, the light-cured material disclosed in the present application has good leveling property. The uncured liquid light-cured material can easily enter the small gap of the mold, which can greatly improve the efficiency and quality of the chip processing by molding method. In addition, the preparation process of the chip is simple, fast, high throughput and low cost, which makes up for the shortcomings of high cost and difficult to quickly mass-produce of the PDMS chamber type digital detection chip, so that the digital detection chip can be more widely applied to the field of biomolecule detection such as nucleic acid and protein.

[0078] The light-cured digital detection chip disclosed in the present application can also be embedded with an anti-evaporation layer, which together with the anti-evaporation channel forms an anti-evaporation system, which can effectively reduce the evaporation loss of the liquid in the chip chamber, and further ensure the smooth progress of the digital detection in the chip. BRIEF DESCRIPTION OF DRAWINGS

[0079] Figure 1 Figure 1 is a structural schematic diagram of the light-cured digital detection chip disclosed in the present application;

[0080] Figure 2 Figure 2 is a mask diagram of the channel structure layer designed in Example 1, wherein Figure A is a first layer mask diagram and Figure B is a second layer mask diagram;

[0081] Figure 3The flow chart of the two preparation processes of the light-cured digital detection chip in the present application is shown in the figure.

[0082] Figure 4 The flow chart of the sample injection of the light-cured digital detection chip in the application example 1 is shown in the figure.

[0083] Figure 5 The micrographs of the light-cured digital detection chip before and after dPCR in the application example 1 are shown in the figure, wherein A is the micrograph of the sample injection of the digital detection reaction solution in the digital detection chip, B is the micrograph of the separation of the oil-separation chamber in the digital detection chip, and C is the micrograph of the digital detection chip after dPCR.

[0084] Figure 6 The micrograph of the light-cured digital detection chip after dPCR in the application test 2 is shown in the figure.

[0085] Figure 7 The micrograph of the light-cured digital detection chip after the sample injection in the application test 3 is shown in the figure, wherein A is the micrograph of the sample distribution channel and the microcavity array, and B is the micrograph of the sample injection port.

[0086] Figure 8 The micrograph of the channel structure layer pattern of the light-cured digital detection chip in the embodiment 2 is shown in the figure.

[0087] Figure 9 The micrograph of the channel structure layer pattern of the light-cured digital detection chip in the embodiment 3 is shown in the figure.

[0088] Figure 10 The micrograph of the sample injection of the digital detection reaction solution of the light-cured digital detection chip in the embodiment 4 is shown in the figure.

[0089] Figure 11 The micrograph of the sample injection port of the light-cured digital detection chip in the embodiment 5 is shown in the figure.

[0090] The figure is:

[0091] 1-chip body, 2-sealing base, 3-fluid channel, 4-microcavity array, 5-mold, 6-hydrophobic substrate, 7-digital detection reaction solution, 8-separation oil, 9-sealing film.

[0092] 11-support layer, 12-evaporation prevention layer, 13-channel structure layer, 14-unhardened channel structure layer light-cured material, 21-sealing layer, 22-base layer, 23-unhardened sealing layer light-cured material, 31-sample distribution channel, 32-evaporation prevention channel.

[0093] 311-sample injection port, 312-tree-shaped channel, 313-branch channel, 314-sample outlet. DETAILED DESCRIPTION

[0094] For further understanding of this application, the following will make specific description to this application in combination with the drawings and examples, but this application is not limited to these examples, and the non-essential improvements and adjustments made by the skilled in the art under the core guiding ideology of this application still belong to the protection scope of this application.

[0095] Figure 1 The light-cured digital detection chip disclosed in this application comprises a chip main body 1 and a sealing base 2.

[0096] The chip main body 1 comprises a support layer 11 and a channel structure layer 13, and an optional anti-evaporation layer 12 according to actual application. The channel structure layer 13 is a thin layer prepared from specific light-cured material, and different channel structures such as fluid channels and micro-chamber arrays are processed on the thin layer for subsequent different digital detection applications. The channel structure can be prepared by first preparing a mold with corresponding channel structure through photolithography, and then preparing the light-cured material with the channel structure through mold turning, i.e. obtaining the channel structure layer 13.

[0097] Figure 2 The mask diagram of the channel structure layer used in Example 1 is given in the middle, and two layers of masks are used because the channel structures prepared in this application are of different heights. The first layer of mask in Figure A comprises fluid channels 3 and micro-chamber arrays 4; the micro-chamber arrays 4 are used to carry the digital detection reaction solution after distribution, and in order to ensure that the detection reaction solution can be distributed into each micro-chamber, the sample distribution channel 31 is further designed with tree-shaped channels 312 for coarse distribution of the sample and branch channels 313 for fine distribution of the sample, as well as a sample inlet 311 and a sample outlet 314; an anti-evaporation channel 32 is also designed to prevent evaporation of the liquid in the chamber. The second layer of mask in Figure B comprises the micro-chamber arrays 4 on the inside and the anti-evaporation channels 32 on the outside.

[0098] Figure 3 The two preparation process flowcharts of the light-cured digital detection chip disclosed in this application are shown in the middle.

[0099] In process (A):

[0100] (a) placing the uncured channel structure layer light-cured material 14 on the mold 5;

[0101] (b) connecting the support layer 11 with the anti-evaporation layer 12;

[0102] (c) covering the support layer 11 and the anti-evaporation layer 12 prepared in step (b) on the light-cured material 14 in step (a) to make the light-cured material form a thin film;

[0103] (d) after the light-cured material 14 is cured, the channel structure layer 13 is formed and connected with the support layer 11 and the anti-evaporation layer 12 to constitute the chip main body 1 together;

[0104] (e) Turn the chip body 1 in step (d) upside down from the mold 5, and punch a hole at the position of the inlet and outlet port with a puncher;

[0105] (f) Drop the uncured encapsulation layer photo-curing reagent 23 on the surface of the hydrophobic substrate 6;

[0106] (g) Cover the substrate layer 22 on the photo-curing material 23 in step (f) to form a thin film of the photo-curing material;

[0107] (h) After the photo-curing material 23 is cured, the encapsulation layer 21 is formed, together with the substrate layer 22, to form the encapsulation substrate 2, and then the hydrophobic substrate 6 is removed;

[0108] (i) Closely attach the chip body 1 in step (d) to the encapsulation substrate 2 in step (h), and perform photo-bonding under ultraviolet light.

[0109] In process (B):

[0110] (j) Place the uncured channel structure layer photo-curing material 14 on the mold 5;

[0111] (k) Form a thin film of the photo-curing material 14 by spin coating or blade coating;

[0112] (l) Cure the photo-curing material thin film in step (k) to form the channel structure layer 13;

[0113] (m) Connect the anti-evaporation layer 12 on the channel structure layer 13 in step (l);

[0114] (n) Connect the support layer 11 on the anti-evaporation layer 12 in step (m) to form the chip body 1;

[0115] (o) Turn the chip body 1 in step (n) upside down from the mold 5, and punch a hole at the position of the inlet and outlet port with a puncher;

[0116] (p) Drop the uncured encapsulation layer photo-curing reagent 23 on the surface of the hydrophobic substrate 6;

[0117] (q) Cover the substrate layer 22 on the photo-curing material 23 in step (p) to form a thin film of the photo-curing material;

[0118] (r) After the photo-curing material 23 is cured, the encapsulation layer 21 is formed, together with the substrate layer 22, to form the encapsulation substrate 2, and then the hydrophobic substrate 6 is removed;

[0119] (s) Closely attach the chip body 1 in step (o) to the encapsulation substrate 2 in step (r), and perform photo-bonding under ultraviolet light.

[0120] Example 1. Fabrication of thiol-ene polysiloxane based photo-cured digital detection chip

[0121] a) Channel structure layer mold processing: spin-coat photoresist (Microchem, SU-83050) on a clean and dry silicon wafer with a thickness of about 30 μm, pre-bake; place the first layer of mask film (AZ® 5200-17i) on the photoresist, expose for 12 s, post-bake; spin-coat photoresist (Microchem, SU-83050) again on the first layer of mask film with a thickness of about 70 μm, pre-bake; place the second layer of mask film (AZ® 5200-17i) on the single-side photoetching machine, align the first layer of photoresist pattern with the second layer of mask film pattern using the instrument alignment system, expose for 15 s, post-bake; finally develop and hard-bake; before use, modify the mold with 0.5% mass fraction of 1H, 1H, 2H, 2H-perfluorodecyltriethoxysilane dissolved in fluorine oil to facilitate the subsequent chip demolding smoothly. Figure 2 A) tightly on the photoresist, expose for 12 s, post-bake; spin-coat photoresist (Microchem, SU-83050) again on the first layer of mask film with a thickness of about 70 μm, pre-bake; place the second layer of mask film (AZ® 5200-17i) on the single-side photoetching machine, align the first layer of photoresist pattern with the second layer of mask film pattern using the instrument alignment system, expose for 15 s, post-bake; finally develop and hard-bake; before use, modify the mold with 0.5% mass fraction of 1H, 1H, 2H, 2H-perfluorodecyltriethoxysilane dissolved in fluorine oil to facilitate the subsequent chip demolding smoothly. Figure 2 B) on the single-side photoetching machine, align the first layer of photoresist pattern with the second layer of mask film pattern using the instrument alignment system, expose for 15 s, post-bake; finally develop and hard-bake; before use, modify the mold with 0.5% mass fraction of 1H, 1H, 2H, 2H-perfluorodecyltriethoxysilane dissolved in fluorine oil to facilitate the subsequent chip demolding smoothly.

[0122] b) Support layer processing: prepare a solution containing 96.9% polyurethane acrylate, 3% 1,6-hexanediol diacrylate and 0.1% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone by mass percentage; place the solution between two clean release films, cover with a glass plate on top and bottom, and cure into a thin plate with a thickness of about 2 mm under ultraviolet light (365 nm, 45 mW / cm 2 , 60 s) as needed, cut into the specified size, and use as a support layer.

[0123] c) Chip body processing: prepare a photo-cured material containing 24.3% (mercapto) methylsiloxane-dimethylsiloxane copolymer, 74.6% vinyl terminated polydimethylsiloxane, 0.8% 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane and 0.3% photoinitiator (2,4,6-trimethylbenzoyl) diphenyl phosphine oxide by mass percentage, and drop onto the mold processed in step a); then place a vapor-proof film with the same area as the microcavity array region on one side of the support layer prepared in step b) as a vapor-proof layer; cover the support layer on the photo-cured material solution, so that the photo-cured material forms a thin layer and fills the pattern area of the mold; under ultraviolet light (365 nm, 45 mW / cm 2 , 40 s), the photo-cured material is cured to form a channel structure layer, which together with the support layer and the vapor-proof layer constitutes the chip body; flip it off the mold, and finally use a puncher to punch holes at the inlet and outlet positions.

[0124] d) Sealing substrate processing: a thiol-ene solution containing 73.9% pentaerythritol tetra(3-mercaptopropionate), 25.1% triallyl isocyanurate and 1% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone by mass percentage was prepared; the thiol-ene solution was dropped on the hydrophobic substrate, and a glass plate was used as a substrate layer, under UV irradiation (365 nm, 2.5 mW / cm 2 , 30 s), the thiol-ene solution was cured to form a sealing layer, which together with the substrate layer constituted a sealing substrate.

[0125] e) Chip sealing: the chip body of step c) was aligned and closely attached to the sealing substrate of step d), and UV irradiation (365 nm, 45 mW / cm 2 , 60 s) was applied for sealing to form a photocured digital detection chip.

[0126] Application test 1

[0127] A plasmid containing a beta-actin (ACTB) gene was dissolved in nuclease-free water and gradient diluted as a sample solution to be tested; a digital detection reaction solution was prepared, including 30 μL of 2x TaqMan Universal PCR master mix, 21 μL of nuclease-free water, 3 μL of 20x primer / probe reaction, 3 μL of 0.2% Tween-20 aqueous solution by mass fraction, and 3 μL of the sample solution to be tested. (A small amount of pigment was added to the solution for clear and intuitive representation of the liquid change in the chamber.)

[0128] A solution containing 55.2% pentaerythritol tetra(3-mercaptopropionate), 44.3% diallyl maleate and 0.5% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone by mass percentage was prepared as a separation oil.

[0129] The photocured digital detection chip prepared in Example 1 was used for application test, and a positive and negative pressure combination was used for sampling, and the flow chart is shown in Figure 4 , and the specific steps are as follows:

[0130] (a) All sample inlet and outlet ports of the photocured digital detection chip were closed by using the sealing film 9, and the chip was subjected to negative pressure treatment to form a negative pressure environment in the chip;

[0131] (b) The photocured digital detection chip after negative pressure treatment was taken out, the seal of the sample distribution channel sample inlet was broken, and the digital detection reaction solution 7 was injected into the sample distribution channel and the microcavity array in the chip from the sample inlet under external pressure;

[0132] (c) After the sample injection is completed, all the sealing measures of the photo-cured digital detection chip are removed, and the partition oil 8 is injected from the sample dispensing channel injection port to replace the liquid in the sample dispensing channel;

[0133] (d) After the fluid in the flow channel is stabilized, the photo-cured digital detection chip is placed under ultraviolet light, and the partition oil 8 is cured, i.e., the overall injection process is completed.

[0134] Figure 5 After the photo-cured digital detection chip adopts the injection process of Figure 4 , the distribution of the digital detection reaction solution in the chip and the partition of the chambers by the partition oil, and the situation in the chip channel and chambers after dPCR are shown. Figure A shows Figure 4 (b) The overall situation in the chip channel after the step is completed, the digital detection reaction solution completely fills the sample dispensing channel and the micro-chamber array within 30s, and all the gas in the micro-chamber array is discharged. Figure B shows Figure 4 (c) The overall situation in the chip channel after the step is completed, the digital detection reaction solution in the sample dispensing channel is replaced by the partition oil, and each chamber containing the digital detection reaction solution is not connected. Figure C shows the overall situation of the chip after the injection process is completed and after dPCR, in which the partition oil in the sample dispensing channel is well adhered to the channel wall, and there is no obvious gas entry phenomenon, and there is also no obvious solution evaporation phenomenon in the chambers.

[0135] Application Test 2

[0136] The photo-cured digital detection chip prepared in Example 1 is subjected to application test, and the test conditions are basically the same as in Application Test 1, except that the partition oil is replaced by silicone oil, which contains 70% 10cst dimethyl silicone oil, 28% PDMS prepolymer (Momentive, RTV-615) and 2% PDMS curing agent (Momentive, RTV-615) by mass percentage.

[0137] Through testing, although the silicone oil can also separate the digital detection reaction solution in each micro-chamber, the sample dispensing channel gas entry and the solution evaporation in the micro-chamber array phenomenon Figure 6 ) easily occur during dPCR. The possible reason for the analysis is that it does not carry the same photo-cured functional groups as the channel structure layer, so it only relies on its own adhesion to the sample dispensing channel.

[0138] Application Test 3

[0139] The light-cured digital detection chip prepared in Example 1 was subjected to application test, and the test conditions were basically the same as in Application Test 1, except that the separation oil was replaced by a thiol-ene light-cured oil, which contained 41.9% tetrapentaerythritol tetrakis(3-mercaptopropionate), 57.1% triallyl isocyanurate and 1% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone by mass percentage.

[0140] It was found that when the thiol-ene light-cured oil was used, more pressure was needed to press the separation oil into the chip channel during sample injection, which could cause deformation of the sample distribution channel and flow of part of the separation oil into the microcavity array Figure 7 , and also cause excessive pressure at the chip sample inlet and liquid leakage Figure 7 , thus posing a risk of damage to the chip seal.The reason was analyzed. The thiol-ene light-cured oil, although having similar functional groups to the light-cured material of the channel structure layer and could adhere to the channel structure layer, had a relatively high viscosity, thus causing the above-mentioned adverse effects.

[0141] Example 2

[0142] The preparation process was basically the same as in Example 1, except that in step c) during processing of the chip body, no leveling agent was added when the light-cured material was prepared, and the raw material composition was 24.5% (mercapto)methylsiloxane-dimethylsiloxane copolymer, 75.2% vinyl-terminated polydimethylsiloxane and 0.3% photoinitiator (2,4,6-trimethylbenzoyl) diphenyl phosphine oxide.

[0143] It was found that the light-cured material without the leveling agent would have low pattern transfer quality during processing of the chip body, and bubbles Figure 8 would occur at the structure of the channel pattern with fine structures, thus affecting the distribution of the digital detection reaction solution in the chip and the separation process of the separation oil, and posing a risk of mutual interference between the microcavities.

[0144] Example 3

[0145] The preparation process was basically the same as in Example 1, except that in step c) during processing of the chip body, the leveling agent 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane was replaced by an equal amount of silicone acrylate (Wacker, TEGORad 2500).

[0146] It was found that the light-cured material prepared by using the silicone acrylate as the leveling agent would have a torn microcavity array pattern, which could cause communication between two cavities, which might be due to the non-uniform curing of the light-cured material prepared by using this leveling agent during the curing process. Figure 9

[0147] Example 4 Preparation of a photo-cured digital detection chip based on thiol-ene solution

[0148] a) Channel structure layer mold processing: exactly the same as step a) in Example 1.

[0149] b) Channel structure layer processing: a thiol-ene photo-cured material containing 73.9% of pentaerythritol tetra(3-mercaptopropionate), 25.1% of triallyl isocyanurate and 1% of a photoinitiator 2-hydroxy-2-methyl-l-phenyl-l-propanone was prepared by mass percentage; the photo-cured material was spin-coated on the mold processed in step a) with a thickness of about 150 μm, and was irradiated with UV light (365 nm, 45 mW / cm 2 , 5 s) to cure the photo-cured material to form the channel structure layer; then a non-evaporable membrane with the same area as the microchamber array region was attached to the top of the channel structure layer as a non-evaporable layer.

[0150] c) Chip body processing: a solution containing 94.1% of urethane acrylate, 5% of ethoxyethoxyethyl acrylate and 0.9% of a photoinitiator 2-hydroxy-2-methyl-l-phenyl-l-propanone was prepared by mass percentage; the solution was poured on the channel structure layer prepared in step b); and was cured under UV light (365 nm, 45 mW / cm 2 , 60 s) to form a support layer with a thickness of about 2 mm, which together with the channel structure layer and the non-evaporable layer constituted the chip body; the chip body was flipped off from the mold, and finally a puncher was used to punch holes at the positions of the sample inlet and outlet.

[0151] d) Sealing substrate processing: a thiol-ene solution containing 41.9% of pentaerythritol tetra(3-mercaptopropionate), 57.1% of triallyl isocyanurate and 1% of a photoinitiator 2-hydroxy-2-methyl-l-phenyl-l-propanone was prepared by mass percentage; the thiol-ene solution was dropped on the hydrophobic substrate, and a glass plate was used as a substrate layer; under UV light (365 nm, 2.5 mW / cm 2 , 30 s), the thiol-ene solution was cured to form a sealing layer, which together with the substrate layer constituted the sealing substrate.

[0152] e) Chip sealing: the chip body of step c) was aligned and tightly attached to the sealing substrate of step d), and was irradiated with UV light (365 nm, 45 mW / cm 2 , 30 s) to form a photo-cured digital detection chip.

[0153] It was found that the channel structure layer based on thiol-ene photo-curable material was more likely to have defects at the sample distribution channel during the processing than the thiol-ene polysiloxane; meanwhile, the time required for sample injection was about 5-10 times of the thiol-ene polysiloxane based photo-cured digital detection chip, and some gas still existed in the micro-chamber after the sample injection process Figure 10 ). The reason was that the gas permeability of the photo-cured digital detection chip based on thiol-ene photo-curable material was lower than that of the thiol-ene polysiloxane, which would adversely affect the subsequent digital detection process.

[0154] Example 5 Preparation of photo-cured digital detection chip based on perfluoropolyether acrylate

[0155] a) Channel structure layer mold processing: exactly the same as step a) in Example 1.

[0156] b) Channel structure layer processing: prepare photo-curable material containing 98% perfluoropolyether acrylate, 1% silicone polyether acrylate and 1% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone by mass percentage; spin-coat the photo-curable material solution on the mold processed in step a) with a thickness of about 150 μm, apply ultraviolet light (365 nm, 45 mW / cm 2 , 60 s) in a nitrogen environment, and the photo-curable material is cured to form a channel structure layer; then attach an evaporation-resistant film with the same area as the micro-chamber array region to the top of the channel structure layer as an evaporation-resistant layer.

[0157] c) Chip body processing: prepare a solution containing 94.1% polyurethane acrylate, 5% ethoxyethoxy acrylate and 0.9% photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone by mass percentage; pour the solution on the channel structure layer prepared in step b); cure under ultraviolet light (365 nm, 45 mW / cm 2 , 60 s) to form a support layer with a thickness of about 2 mm, which together with the channel structure layer and the evaporation-resistant layer constitutes the chip body; turn it over from the mold, and finally punch holes at the sample inlet and outlet positions with a puncher.

[0158] d) Sealing substrate processing: take a plastic plate with an area greater than or equal to that of the chip body as the substrate layer, and bond an adhesive tape with acrylate functional groups to the plastic plate as the sealing layer, which together constitute the sealing substrate.

[0159] e) Chip sealing: align and tightly attach the chip body of step c) with the sealing substrate of step d), and apply ultraviolet light (365 nm, 45 mW / cm 2 , 90 s) in a nitrogen environment for sealing to form a photo-cured digital detection chip.

[0160] Tests showed that channel structure layers based on perfluoropolyether acrylates are more prone to cracking during processing compared to thiol-olefin polysiloxanes. Figure 11 Therefore, its injection port is more prone to leakage problems; at the same time, the production cost of perfluoropolyether acrylate is relatively high, which limits its application scenarios.

Claims

1. A method for detecting a photo-cured digital detection chip, characterized in that, The light-cured digital detection chip comprises a chip body and a sealing base; The chip body comprises a support layer and a channel structure layer; The channel structure layer is a thin layer prepared by light-curing of a light-cured material, and the thin layer is processed with a channel structure; The channel structure layer comprises a fluid channel for fluid flow and a microcavity array for accommodating samples; The fluid channel comprises a sample distribution channel and an anti-evaporation channel; The sample distribution channel comprises a tree-shaped channel, several branch channels, a sample inlet and a sample outlet; The anti-evaporation channel surrounds the sample distribution channel region and the microcavity array region; The light-cured material comprises a raw material containing a light-cured functional group and a photoinitiator; The raw material containing a light-cured functional group is selected from mercapto-containing polysiloxane and alkenyl-containing polysiloxane; The light-cured material has air permeability after light-curing; The light-cured material further comprises a leveling agent selected from 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane; The detection method comprises: (1) rapidly distributing the digital detection reaction solution into the microcavity array in the channel structure by driving with negative pressure and / or externally applied pressure, then injecting separation oil and replacing the liquid in the sample distribution channel in the channel structure after the sample is completely injected, and then performing light-curing until the separation oil is cured to complete the sample injection process after the fluid is stable; The separation oil comprises tetra(3-mercaptopropionic acid) pentaerythritol ester and maleic acid diallyl ester; (2) placing the digital detection chip on a hot plate, setting the temperature rising and falling program of the hot plate according to the digital detection reaction to be taken, and performing the digital detection reaction; after the digital detection reaction is completed, the chip is placed under a fluorescence field, and the fluorescence image of the microcavity array of the chip is collected.

2. The detection method of the light-cured digital detection chip according to claim 1, wherein: The thin layer prepared by light-curing has a reticular structure, and the mesh holes are selected from mesopores and micropores with a pore size of not more than 20 nm.

3. The detection method of the light-cured digital detection chip according to claim 1, wherein: An anti-evaporation layer is further arranged between the support layer and the channel structure layer, and the anti-evaporation layer is located above the channel structure region in the channel structure layer and has an area greater than or equal to the area of the channel structure region; The anti-evaporation layer is an air-tight film and is fixedly connected with the support layer and the channel structure layer, respectively.

4. The detection method of the light-cured digital detection chip according to claim 1, wherein: The material of the support layer is selected from one or more of glass, plastic, rubber and light-cured material; The sealing base can be a single base layer or a composite layer composed of a sealing layer and a base layer, and the material is selected from one or more of glass, plastic, rubber, polydimethylsiloxane, double-sided adhesive tape and light-cured material.

5. Application of the detection method of the light-cured digital detection chip according to any one of claims 1 to 4 in the field of biomolecular detection.

6. Application of the detection method of the light-cured digital detection chip according to any one of claims 1 to 4 in the field of cell culture or microorganism culture.

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