阅读说明：本技术 多层微流控芯片封装器件、多层微流控芯片及其应用 (Multilayer microfluidic chip packaging device, multilayer microfluidic chip and application thereof ) 是由 张秀莉 罗勇 邓权锋 于 2021-03-31 设计创作，主要内容包括：本发明涉及一种多层微流控芯片封装器件,包括第一基板和第二基板,第一基板设置有凸部,第二基板设置有与凸部配合连接的凹部,凸部连接凹部的不同位置,以调整第一基板和第二基板之间的距离；多层基板包括弹性材质的第三基板,第三基板设置于第一基板与第二基板之间,且第三基板的厚度大于第一基板与第二基板之间的距离,以使第三基板在第一基板与第二基板的压合下产生弹性变形。本发明通过凸部在凹部内的移动切换有效调整并控制基板之间的距离,从而实现基板之间的紧密连接,相较于现有技术而言,本发明无需压力产生装置,显著减小了芯片体积,而且易于通过注塑及激光雕刻等方法进行多层基板的标准化生产。(The invention relates to a multilayer microfluidic chip packaging device which comprises a first substrate and a second substrate, wherein the first substrate is provided with a convex part, the second substrate is provided with a concave part which is matched and connected with the convex part, and the convex part is connected with different positions of the concave part so as to adjust the distance between the first substrate and the second substrate; the multilayer substrate comprises a third substrate made of elastic materials, the third substrate is arranged between the first substrate and the second substrate, and the thickness of the third substrate is larger than the distance between the first substrate and the second substrate, so that the third substrate generates elastic deformation under the pressing of the first substrate and the second substrate. Compared with the prior art, the invention does not need a pressure generating device, obviously reduces the volume of a chip, and is easy to carry out standardized production of multilayer substrates by methods such as injection molding, laser engraving and the like.)

1. A multilayer microfluidic chip packaging device is characterized in that: the multilayer substrate comprises a plurality of layers of substrates, wherein fluid channels are arranged on part of the substrates in the plurality of layers of substrates;

the multilayer substrate comprises a first substrate and a second substrate, wherein the first substrate is provided with a convex part, the second substrate is provided with a concave part matched and connected with the convex part, and the convex part is connected with different positions of the concave part so as to adjust the distance between the first substrate and the second substrate;

the multilayer substrate comprises a third substrate made of elastic materials, the third substrate is arranged between the first substrate and the second substrate, and the thickness of the third substrate is larger than the distance between the first substrate and the second substrate, so that the third substrate generates elastic deformation under the pressing of the first substrate and the second substrate, and the first substrate, the third substrate, the second substrate and the third substrate are tightly attached.

2. The multi-layer microfluidic chip packaging device of claim 1, wherein: the concave part comprises a plurality of teeth, a gear is arranged between every two adjacent teeth, and the convex part can be clamped on the gear.

3. The multi-layer microfluidic chip packaging device of claim 1, wherein: the convex part and/or the concave part are/is made of hard materials capable of generating slight deformation, so that the convex part can move and switch on the gear position of the concave part.

4. The multi-layer microfluidic chip packaging device of claim 1, wherein: the display device further comprises a porous membrane, wherein the porous membrane is arranged between the first substrate and the second substrate.

5. A multilayer microfluidic chip, characterized in that: a multi-layer microfluidic chip package device comprising the multi-layer microfluidic chip package device of any one of claims 1-4;

the substrate is provided with a fluid channel, the substrate is provided with a plurality of fluid channels, and the substrate is provided with a plurality of normally-closed valves.

6. The multi-layer microfluidic chip of claim 5, wherein: the base plate is provided with holes for containing test solution, the holes are connected with normally-closed valves through fluid channels, and the normally-closed valves control the transfer of the test solution among the holes through the fluid channels.

7. The multi-layer microfluidic chip of claim 6, wherein: the holes comprise a hole a, a hole b and a hole c, and the volume of the hole b and/or the hole c is larger than that of the hole a; the fluid channel comprises a fluid channel a, a fluid channel b and a fluid channel c, and the hole a, the hole b and the hole c are respectively connected with the normally-closed valve through the fluid channel a, the fluid channel b and the fluid channel c.

8. The multi-layer microfluidic chip of claim 7, wherein: the fluid channel also comprises a fluid channel d, and the fluid channel d is connected with normally closed valves which are respectively connected with the fluid channel a and the fluid channel b; the fluid channel further comprises a fluid channel e, and the fluid channel e is connected with normally closed valves which are respectively connected with the fluid channel a and the fluid channel c.

9. The multi-layer microfluidic chip of claim 7, wherein: the holes a are arranged side by side on the substrate in a row, one hole a in each row is divided into a plurality of chambers by the porous membrane along the vertical direction, and one chamber is provided with a biochemical reaction carrier.

10. Use of the multi-layered microfluidic chip according to claim 5 for immunoassay.

Technical Field

The invention relates to the technical field of microfluidic chips, in particular to a multilayer microfluidic chip packaging device, a multilayer microfluidic chip and application thereof.

Background

Microfluidics is a technology for controlling fluid in a microscale channel, layers of a multilayer microfluidic chip applying the microfluidics need to be tightly attached, and the purpose of tight attachment is to prevent a test solution from leaking from gaps between the layers. At present, the attaching mode of the multilayer micro-fluidic chip mainly comprises the following steps: 1) thermal bonding, which is mainly used for materials such as glass, silicon, hard plastics and the like, has multiple steps and complex operation and needs professional skills; 2) plasma sealing, but the method is limited to a few limited materials such as PDMS and the like, and has low efficiency, poor repeatability and difficulty in industrial production; 3) the layer is tightly attached to the layer by means of pressure attachment, the mode usually uses the cooperation of elastic materials and hard materials, such as a PDMS elastic layer and a PMMA plate, and utilizes the cooperation of screws and nuts to apply pressure, so that the elastic layer generates some deformation, and the sealing effect is achieved by extrusion. Thus, in general, the microfluidic chip packages of the prior art lack a simple, reliable, and standardized substrate attachment design.

Disclosure of Invention

Therefore, the technical problem to be solved by the present invention is to overcome the defect that the microfluidic chip package in the prior art lacks a simple, reliable and standardized substrate attachment design.

In order to solve the above technical problems, a first aspect of the present invention provides a multilayer microfluidic chip package device, including a multilayer substrate, wherein a part of the multilayer substrate is provided with a fluid channel; (ii) a

The multilayer substrate comprises a first substrate and a second substrate, wherein the first substrate is provided with a convex part, the second substrate is provided with a concave part matched and connected with the convex part, and the convex part is connected with different positions of the concave part so as to adjust the distance between the first substrate and the second substrate; (ii) a

The multilayer substrate comprises a third substrate made of elastic materials, the third substrate is arranged between the first substrate and the second substrate, and the thickness of the third substrate is larger than the distance between the first substrate and the second substrate, so that the third substrate generates elastic deformation under the pressing of the first substrate and the second substrate, and the first substrate, the third substrate, the second substrate and the third substrate are tightly attached.

In one embodiment of the invention, the concave part comprises a plurality of teeth, a shift position is arranged between every two adjacent teeth, and the convex part can be clamped on the shift position.

In one embodiment of the present invention, the convex portion and/or the concave portion is made of a hard material that can be slightly deformed so that the convex portion can be moved and switched in the shift position of the concave portion.

In one embodiment of the present invention, further comprising a porous membrane disposed between the first substrate and the second substrate.

The invention provides a multilayer microfluidic chip in a second aspect, which comprises the multilayer microfluidic chip packaging device;

the substrate is provided with a fluid channel, the substrate is provided with a plurality of fluid channels, and the substrate is provided with a plurality of normally-closed valves.

In one embodiment of the invention, the substrate is provided with holes for containing the test solution, and the holes are connected with normally-closed valves through fluid channels, and the normally-closed valves control the transfer of the test solution among the holes through the fluid channels.

In one embodiment of the invention, the pores include pore a, pore b and pore c, the volume of pore b and/or pore c is greater than the volume of pore a; the fluid channel comprises a fluid channel a, a fluid channel b and a fluid channel c, and the hole a, the hole b and the hole c are respectively connected with the normally-closed valve through the fluid channel a, the fluid channel b and the fluid channel c.

In one embodiment of the invention, the fluid channel further comprises a fluid channel d, and the fluid channel d is connected with normally closed valves which are respectively connected with the fluid channel a and the fluid channel b; the fluid channel further comprises a fluid channel e, and the fluid channel e is connected with normally closed valves which are respectively connected with the fluid channel a and the fluid channel c.

In one embodiment of the present invention, the holes a are arranged side by side in a row on the substrate, one of the holes a in each row is divided into a plurality of chambers by the porous membrane in a vertical direction, and one of the chambers is provided with a biochemical reaction carrier.

The third aspect of the invention provides an application of the multilayer microfluidic chip in immunoassay.

Compared with the prior art, the technical scheme of the invention has the following advantages:

compared with the prior art, the invention does not need a pressure generating device, obviously reduces the volume of the chip, makes the chip more beautiful, and is easy to carry out the standardized production of the multilayer substrate by methods of injection molding, laser engraving and the like.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

Fig. 1 is a schematic structural diagram of a first embodiment of the present invention.

Fig. 2 is a first schematic diagram illustrating a substrate bonding principle according to a first embodiment of the invention.

Fig. 3 is a schematic diagram illustrating a substrate bonding principle in the first embodiment of the invention.

Fig. 4 is a first schematic structural diagram of a third embodiment of the present invention.

Fig. 5 is a schematic structural diagram of a third embodiment of the present invention.

Fig. 6 is a schematic diagram of a normally-closed valve in an open state according to a third embodiment of the present invention.

Fig. 7 is a schematic diagram of a normally closed valve in a closed state according to a third embodiment of the present invention.

Fig. 8 is a schematic structural diagram of a multilayer microfluidic chip in four to seven embodiments of the present invention.

FIG. 9 is a graph showing a standard curve of AMH detection in the fourth embodiment of the present invention.

FIG. 10 is a schematic diagram of a standard curve for the detection of the inflammatory factor IL-6 in example six of the present invention.

The specification reference numbers indicate: 110. a first substrate; 120. a second substrate; 130. a third substrate; 140. a convex portion; 150. a recess; 151. a gear position; 152. teeth; 160. a porous membrane; 170. a fluid channel;

210. an upper hard substrate; 220. an intermediate layer elastic film; 230. a lower hard substrate;

311. a hole a; 312. a hole b; 313. a hole c; 321. a fluid channel a; 322. a fluid channel b; 323. a fluid channel c; 324. a fluid channel d; 325. a fluid channel e; 330. a chamber; 340. and (3) biochemical reaction carriers.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example one

The first embodiment of the multilayer microfluidic chip packaging device provided by the present invention is described below, and referring to fig. 1 to 3, the first embodiment includes:

referring to fig. 1 to 3, a multilayer microfluidic chip package device includes a multilayer substrate, a part of the multilayer substrate is provided with a fluid channel 170, the multilayer substrate includes a first substrate 110 and a second substrate 120, a porous membrane 160 is disposed between the first substrate 110 and the second substrate 120, the first substrate 110 is provided with a convex portion 140, and the second substrate 120 is provided with a concave portion 150 in mating connection with the convex portion 140. Preferably, the first substrate 110 and the second substrate 120 are rigid substrates, the protrusion 140 is similar to a male head structure, the recess 150 is similar to a female head structure, and the protruding direction of the male head and the recess direction of the female head are both perpendicular to the surfaces of the substrates, so that the male head can be embedded into the female head when the package is attached, and thus the distance between the first substrate 110 where the male head is located and the second substrate 120 where the female head is located can be fixed. Here, the first substrate 110 and the second substrate 120 may be hard substrates.

Further, the multilayer substrate includes a third substrate 130, the third substrate 130 is an elastic substrate, the third substrate 130 is disposed between the first substrate 110 and the second substrate 120, and the thickness of the third substrate 130 is greater than the distance between the first substrate 110 and the second substrate 120, so that the third substrate 130 is elastically deformed under the pressing of the first substrate 110 and the second substrate 120, and the first substrate 110 and the third substrate 130, and the second substrate 120 and the third substrate 130 are tightly attached to each other, thereby achieving the sealing of the upper fluid channel 170.

In this embodiment, the third substrate 130 may be a composite substrate, the composite substrate is formed by stacking a plurality of sub-substrates, some of the sub-substrates are made of elastic material, some of the sub-substrates are made of hard material, the topmost sub-substrate and the bottommost sub-substrate are made of elastic material, and the sub-substrates directly bonded to the hard sub-substrates are made of elastic material. Thus, when the first substrate 110 and the second substrate 120 are packaged and bonded, all the elastic sub-substrates in the composite substrate are elastically deformed under the pressing of the first substrate 110 and the second substrate 120, so that the elastic sub-substrates in the composite substrate are tightly bonded with the upper and lower sub-substrates or the first substrate 110 or the second substrate 120 directly bonded with the elastic sub-substrates. Of course, the third substrate 130 may be formed of a single elastic sub-substrate, and the invention is not limited thereto.

With continued reference to fig. 2 and fig. 3, preferably, the female head of the present embodiment is designed as a tooth-shaped structure, which specifically includes a plurality of teeth 152, and a shift position 151 is located between two adjacent teeth 152. The male head is designed to be of a T-shaped structure, and specifically comprises a protrusion matched with the gear 151 of the female head, and the protrusion can be clamped on the gear 151, so that the first substrate 110 and the second substrate 120 are attached and positioned.

Further, the material of the male and/or female head is a hard material capable of undergoing slight deformation, such as a hard plastic capable of undergoing slight deformation, for example, Polycarbonate (PC), a blend of polycarbonate and acrylonitrile-butadiene-styrene terpolymer (PC/ABS), Polyamide (PA), Polyetheretherketone (PEEK), Polyketone (POK), Polyoxymethylene (POM), and the like), so that the protrusion of the male head can be moved and switched on the gear position 151 of the female head, thereby adjusting the distance between the first substrate 110 on which the male head is located and the second substrate 120 on which the female head is located. Specifically, the protrusion of the male tab exerts a force on the teeth 152 when moving, so that the teeth 152 contract and deform in a direction away from the protrusion, so that the protrusion slides from the previous shift position 151 to the next shift position 151, and after the force applied to the teeth 152 disappears, the teeth 152 reset, so that the protrusion of the male tab is locked in the shift position 151.

Above-mentioned embodiment is through the effective adjustment of the cooperation of female head and public head and control the distance between the base plate to realize the zonulae occludens between the base plate, compare in prior art, this embodiment need not pressure and produces the device, is showing and is reducing the chip volume, makes the chip more pleasing to the eye, easily carries out the standardized production of multilayer substrate through methods such as moulding plastics and laser sculpture moreover.

Example two

The second embodiment of the multilayer microfluidic chip packaging device provided by the invention is described below, and the second embodiment is realized based on the first embodiment and is expanded to a certain extent on the basis of the first embodiment.

The difference between the second embodiment and the first embodiment is:

in the second embodiment, in order to improve the problem of the wear of the projections. The tip of the tooth in this embodiment may be designed to be circular arc, so that the circular arc-shaped tooth 152 can reduce the abrasion of the protrusion when the protrusion slides.

In a further improvement, the teeth 152 are further provided with two slots, and the two slots are smoothly connected with the arc-shaped tip part. Specifically, the protruding effect that can exert to tooth 152 when removing of public head for the protruding draw-in groove that breaks away from on the tooth 152 and slide to the point portion of tooth 152, at this moment tooth 152 is to keeping away from bellied direction shrink deformation, so that protruding from last gear 151 slides to next gear 151, until protruding card be located the draw-in groove between tooth 152 can, the point portion of tooth 152 resets simultaneously, so that the protruding screens of public head is located between tooth 152, protruding card is located the draw-in groove between tooth 152 promptly, this draw-in groove is used for further improving the stability that protruding card is located between tooth 152.

The second embodiment is an improvement made on the basis of the first embodiment in order to improve the stability of the pin position, but the invention may have other improvements, and the invention is not limited to this.

EXAMPLE III

The third embodiment of the multilayer microfluidic chip provided by the present invention is described below with reference to fig. 4 to 7, and the third embodiment includes:

referring to fig. 4 to 7, the multi-layer microfluidic chip of the present embodiment includes the multi-layer microfluidic chip package device of the above embodiment and a normally-closed valve, the normally-closed valve is connected to the fluid channel 170 on the substrate, and the normally-closed valve is connected to different fluid channels 170 on the substrate in an open state.

In a preferred embodiment of this embodiment, the multilayer microfluidic chip package device is formed by stacking 4 layers of hard substrates and 3 layers of elastic substrates in the first embodiment using 3 sets of male and female head structures in the first embodiment, and the specific content of the multilayer microfluidic chip package device has been described in detail in the foregoing embodiments, which is not described herein again. In this embodiment, the 4 layers of hard substrates are, from top to bottom, a first hard substrate, a second hard substrate, a third hard substrate, and a fourth hard substrate, where a fluid channel is disposed on the fourth hard substrate, and the fluid channel is connected to the normally-closed valve.

The processing of normally closed valves involves the close attachment of an elastic membrane and a chip substrate, and in a laboratory, a common means is dovetail clamping, so that the volume of a chip is increased, the attractiveness of the whole chip is influenced, and the repeatability and reliability of the whole analysis are influenced due to the fact that the pressure of the dovetail clamp is uncontrollable. Normally closed valves are therefore not practical and do not take advantage of fluid control. Therefore, the present invention provides a normally closed valve which is simple, reliable and can be standardized, and its specific structure please refer to fig. 6 and fig. 7, which includes an upper hard substrate 210 with a fluid channel 170 (here, the above fourth hard substrate), an intermediate elastic membrane 220, a lower hard substrate 230 with a pressure channel, the upper hard substrate 210 is provided with the male head described in the above embodiments, and the lower hard substrate 230 is provided with the female head described in the above embodiments, and its specific connection manner is described with reference to the above embodiments. In operation, when the substrate is under normal pressure, the fluid channel 170 of the upper hard substrate 210 is not connected, and fluid cannot flow, as shown in detail in fig. 6; when the pressure is negative, the middle layer elastic membrane 220 is sucked into the pressure channel of the lower layer, and the fluid channel 170 of the upper layer hard substrate 210 becomes connected, so that the fluid flow can be performed, as shown in detail in fig. 7.

The substrate of the chip is provided with holes for containing the test solution, the holes are connected with a normally-closed valve through a fluid channel 170, and the normally-closed valve controls the transfer of the test solution among the holes through the fluid channel 170. Preferably, the shape of the hole on the chip substrate can be various shapes such as a column, a cylinder, a cone, a big cylinder connected with a small cylinder and the like; the holes can be light-transmitting or light-proof; the volume of each hole is 1-1000 microliter. The test solution in the hole can be enzyme-labeled secondary antibody, fluorescence-labeled secondary antibody, Raman-labeled secondary antibody, cleaning solution, substrate, stop solution, oxygen supply body, chemiluminescence test solution and the like.

Continuing to refer to fig. 4 and fig. 5, specifically, the holes include a hole a311, a hole b312, and a hole c313, where the holes a311 are arranged side by side on the substrate in a row, one of the holes a311 in each row is partitioned into a plurality of chambers 330 by the porous membrane 160 along a vertical direction, one of the chambers 330 is provided with a biochemical reaction carrier 340, and the biochemical reaction carrier 340 may be a porous material such as magnetic beads, microbeads, nanoparticles, polyurethane foam, paper, MOF, and the like. In a preferred embodiment, the volume of the holes b312 and c313 is greater than the volume of the hole a 311.

The fluid channel 170 includes a fluid channel 170a, a fluid channel 170b, a fluid channel 170c, a fluid channel 170d, and a fluid channel 170e, the hole a311, the hole b312, and the hole c313 are connected to the normally closed valves through the fluid channel 170a, the fluid channel 170b, and the fluid channel 170c, respectively, the fluid channel 170d is connected to the normally closed valves connecting the fluid channel 170a and the fluid channel 170b, respectively, and the fluid channel 170e is connected to the normally closed valves connecting the fluid channel 170a and the fluid channel 170c, respectively. In operation, the normally closed valve controls the fluid passage 170 to be open or closed, thereby controlling the transfer of the test solution between the wells.

Example four

The application example four of the multilayer microfluidic chip provided by the invention in the aspect of immunoassay is described below, and the example four is realized based on the example three and is expanded to a certain extent on the basis of the example three.

The embodiment provides an application of a multilayer microfluidic chip in detecting AMH, the specific structure of the chip, the marks of the holes and the marks of the normally-closed valves are shown in fig. 8, the chip is formed by overlapping 5 layers of hard substrates and 4 layers of elastic substrates, the hard substrates are made of PC/ABS, the elastic substrates are made of PDMS, and the 4 sets of male-female head locking structures in the embodiment are used. The pore diameter of the porous membrane 160 is 10 micrometers, the immune carrier is 100 micrometers of glass beads, primary antibodies for AMH pairs are modified on the surfaces of the glass beads, cleaning liquid is contained in a hole 017, a substrate is contained in a hole 018, an oxygen supply body is contained in a hole 019, and stop solution is contained in a hole 020; an enzyme-labeled secondary antibody of AMH is contained in the hole A1-A8, a patient serum sample is also contained in the hole B1-B3, AMH standard solutions with different concentrations are contained in the hole B4-B8, the volume of each hole is 140 microliters, and the transfer of the test solution among the holes on the chip is controlled through a normally closed valve.

The method for detecting AMH by using the multilayer microfluidic chip comprises the following steps:

the first step is as follows: incubation

The sample in the hole B1-B8 is firstly kept still and incubated for 1 hour, and then the AMH molecules are combined with the primary antibody on the surface of the glass beads, so that the AMH molecules are fixed on the surface of the glass beads;

the second step is that: matrix solution removal

By controlling normally closed valves 010, 011 and 013, the matrix solutions in wells B1-B8 were all flowed into individual wells 11, 21, 31 and 41;

the third step: cleaning of

The cleaning solution in the holes 017 is evenly distributed into B1-B8 by controlling the normally closed valves 004 and 006, after a certain period of cleaning, the cleaning solution in the holes B1-B8 is totally flowed into the individual holes 11, 21, 31 and 41 by controlling the normally closed valves 010, 011 and 013, and the cleaning step is repeated three times;

the fourth step: coupled enzyme-labeled secondary antibody

By controlling a normally-closed valve 007-009, an enzyme-labeled secondary antibody solution in the hole A1-A8 flows into the hole B1-B8, and is incubated for a period of time, so that the enzyme-labeled secondary antibody is combined with AMH on the wall of the glass bead;

the fifth step: matrix solution removal

By controlling normally closed valves 010, 011 and 013, the matrix solutions in wells B1-B8 were all flowed into individual wells 11, 21, 31 and 41;

and a sixth step: cleaning of

The cleaning solution in the holes 017 is evenly distributed into B1-B8 by controlling the normally closed valves 004 and 006, after a certain period of cleaning, the cleaning solution in the holes B1-B8 is totally flowed into the individual holes 11, 21, 31 and 41 by controlling the normally closed valves 010, 011 and 013, and the cleaning step is repeated three times;

the seventh step: enzyme-catalyzed substrates

By controlling normally closed valves 002, 003, 005 and 006, the substrate and oxygen donor in wells 018 and 019 were equally distributed into wells B1-B8, and incubation for a while, the enzyme on the surface of the glass beads catalyzed the substrate solution to develop color;

eighth step: termination of the reaction

By controlling normally closed valves 001, 005 and 006, the stop solution in the hole 020 is evenly distributed into B1-B8, and the enzyme catalysis substrate reaction is interrupted;

the ninth step: detection of

The standard curve chart of the AMH detection is shown in FIG. 9 by controlling the normally closed valves 010-012, making the reaction liquid in the holes B1-B8 flow into the holes C1-C8, and detecting by a color development method.

According to the embodiment, the AMH content of three patient samples is automatically and quantitatively detected, the result is obtained within 2 hours, because the sample volume is large, and the microbead is used for signal amplification, the result accuracy is higher than that of the traditional 96-well plate, and the cost is lower.

EXAMPLE five

The application example five of the multilayer microfluidic chip provided by the invention in the aspect of immunodetection is introduced below, and the example five is realized based on the example four and is expanded to a certain extent on the basis of the example four.

The microfluidic chip in this embodiment is designed and operated as in the fourth embodiment, except that the wells B1-B8 contain 8 patient samples, so that the microfluidic chip can realize the automatic quantitative detection of the AMH content in the 8 patient samples.

EXAMPLE six

Next, an application example six of the multilayer microfluidic chip provided by the invention in the aspect of immunoassay is described, and the example six is realized based on the above example three and is expanded to a certain extent on the basis of the example three.

The embodiment provides an application of a multilayer microfluidic chip in inflammatory factor detection, wherein the specific structure of the chip, the marks of the holes and the marks of the normally-closed valves are shown in fig. 8, the chip is formed by overlapping 5 layers of hard substrates and 4 layers of elastic substrates, the hard substrates are made of PC, and the elastic substrates are made of PDMS. The pore diameter of the porous membrane 160 is 10 microns, the immune carrier is 200 microns of silica gel particles, the surfaces of the silica gel particles are modified with primary antibodies corresponding to inflammatory factors IL6, cleaning liquid is contained in the hole 017, substrates are contained in the hole 018, oxygen supply bodies are contained in the hole 019, and stop solution is contained in the hole 020. An enzyme-labeled secondary antibody of IL6 is contained in the A1-A8, a clinical sample is also contained in the B1-B3, and IL6 standard substances with different concentrations are also contained in the B4-B8. The volume of each well is 140 microliters, and the transfer of the test solution between the wells on the chip is controlled by a normally closed valve.

The method for detecting the immune factors by using the multilayer microfluidic chip comprises the following steps:

the first step is as follows: incubation

The sample in the hole B1-B8 is incubated for 1 hour at rest, and IL6 molecules are combined with primary antibodies on the surface of the silica gel particles, so that the samples are fixed on the surface of the silica gel particles

The second step is that: matrix solution removal

By controlling normally closed valves 010, 011 and 013, the matrix solutions in wells B1-B8 were all flowed into individual wells 11, 21, 31 and 41;

the third step: cleaning of

The cleaning solution in the holes 017 is evenly distributed into B1-B8 by controlling the normally closed valves 004 and 006, after a certain period of cleaning, the cleaning solution in the holes B1-B8 is totally flowed into the individual holes 11, 21, 31 and 41 by controlling the normally closed valves 010, 011 and 013, and the cleaning step is repeated three times;

the fourth step: coupled enzyme-labeled secondary antibody

By controlling a normally-closed valve 007-009, an enzyme-labeled secondary antibody solution in the hole A1-A8 flows into the hole B1-B8, and is incubated for a period of time, so that the enzyme-labeled secondary antibody is combined with the IL6 on the surface of the silica gel particles;

the fifth step: matrix solution removal

By controlling normally closed valves 010, 011 and 013, the matrix solutions in wells B1-B8 were all flowed into individual wells 11, 21, 31 and 41;

and a sixth step: cleaning of

The cleaning solution in the holes 017 is evenly distributed into B1-B8 by controlling the normally closed valves 004 and 006, after a certain period of cleaning, the cleaning solution in the holes B1-B8 is totally flowed into the individual holes 11, 21, 31 and 41 by controlling the normally closed valves 010, 011 and 013, and the cleaning step is repeated three times;

the seventh step: enzyme-catalyzed substrates

By controlling normally-closed valves 002, 003, 005 and 006, the substrates and oxygen donors in the holes 018 and 019 are evenly distributed into the holes B1-B8, and the enzyme on the surface of the silica gel particles catalyzes the substrate solution for a certain period of time, so that color development is realized;

eighth step: termination of the reaction

By controlling normally closed valves 001, 005 and 006, the stop solution in the hole 020 is evenly distributed into B1-B8, and the enzyme catalysis substrate reaction is interrupted;

the ninth step: detection of

The standard curve chart of IL6 detection is shown in FIG. 10, which is obtained by controlling normally-closed valves 010-012 to make the reaction liquid in the holes B1-B8 flow into the holes C1-C8 and detecting the reaction liquid by a color development method.

The embodiment realizes the automatic quantitative detection of the IL6 content of three patient samples, and the result is obtained within 2 hours, because the sample volume is large, and the result accuracy is higher than that of the traditional 96-well plate by using silica gel particles to amplify the signals, and the cost is lower.

EXAMPLE seven

The seventh embodiment of the application of the multilayer microfluidic chip in the aspect of immunoassay provided by the invention is described below, and the seventh embodiment is realized based on the third embodiment and is expanded to a certain extent on the basis of the third embodiment.

This example provides the application of a multilayer microfluidic chip in the detection of inflammatory factor storm, and the specific structure of the chip, the marks of the wells and the marks of the normally closed valves are shown in fig. 8. The chip is formed by superposing 5 layers of hard substrates and 4 layers of elastic substrates, wherein the hard substrates are made of PC, and the elastic substrates are made of PDMS. The pore diameter of the porous membrane 160 is 10 micrometers, the immune carrier is 200 micrometers of silica gel particles, primary antibodies corresponding to 8 inflammatory factors (IFN-gamma, TNF-alpha, IL-2, IL-6, IL-1 beta, IL-120p70, IL-4 and IL-10) are modified on the surfaces of the silica gel particles, cleaning fluid is contained in a hole 017, a substrate is contained in a hole 018, an oxygen supply body is contained in a hole 019, and stop solution is contained in a hole 020. The same clinical sample is also contained in the holes B1-B8, and enzyme-labeled secondary antibodies corresponding to 8 inflammatory factors are contained in the holes A1-A8.

The method for detecting the immune factor storm by using the multilayer microfluidic chip comprises the following steps:

the first step is as follows: incubation

The sample in the well B1-B8 is incubated for 1 hour at rest, and the inflammatory factor is bound with the primary antibody on the surface of the silica gel particles, so that the inflammatory factor is fixed on the surface of the silica gel particles

The second step is that: matrix solution removal

By controlling normally closed valves 010, 011 and 013, the matrix solutions in wells B1-B8 were all flowed into individual wells 11, 21, 31 and 41;

the third step: cleaning of

The cleaning solution in the holes 017 is evenly distributed into B1-B8 by controlling the normally closed valves 004 and 006, after a certain period of cleaning, the cleaning solution in the holes B1-B8 is totally flowed into the individual holes 11, 21, 31 and 41 by controlling the normally closed valves 010, 011 and 013, and the cleaning step is repeated three times;

the fourth step: coupled enzyme-labeled secondary antibody

By controlling the normally-closed valve 007-009, the enzyme-labeled secondary antibody solution in the hole A1-A8 flows into the hole B1-B8, and is incubated for a period of time, so that the enzyme-labeled secondary antibody is combined with inflammatory factors on the surfaces of the silica gel particles;

the fifth step: matrix solution removal

By controlling normally closed valves 010, 011 and 013, the matrix solutions in wells B1-B8 were all flowed into individual wells 11, 21, 31 and 41;

and a sixth step: cleaning of

The cleaning solution in the holes 017 is evenly distributed into B1-B8 by controlling the normally closed valves 004 and 006, after a certain period of cleaning, the cleaning solution in the holes B1-B8 is totally flowed into the individual holes 11, 21, 31 and 41 by controlling the normally closed valves 010, 011 and 013, and the cleaning step is repeated three times;

the seventh step: enzyme-catalyzed substrates

By controlling normally-closed valves 002, 003, 005 and 006, the substrates and oxygen donors in the holes 018 and 019 are evenly distributed into the holes B1-B8, and the enzyme on the surface of the silica gel particles catalyzes the substrate solution for a certain period of time, so that color development is realized;

eighth step: termination of the reaction

By controlling normally closed valves 001, 005 and 006, the stop solution in the hole 020 is evenly distributed into B1-B8, and the enzyme catalysis substrate reaction is interrupted;

the ninth step: detection of

The reaction solution in the well B1-B8 is flowed into the well C1-C8 by controlling the normally closed valve 010-012, and the detection is carried out by a color development method.

The bedside automatic detection method for the inflammatory factor storm of the critically ill patient can realize bedside automatic detection (8 inflammatory factors), the result is obtained within 2 hours, and due to the fact that the sample size is large, and the silica gel particles are used for signal amplification, the result accuracy is higher than the detection accuracy of a traditional 96-well plate, and the cost is lower.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.