CN112774744A - Micro-fluidic device for multi-target quantitative detection - Google Patents
Micro-fluidic device for multi-target quantitative detection Download PDFInfo
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Abstract
The invention discloses a micro-fluidic device for multi-target quantitative detection in the field of medical immune in-vitro diagnosis, which comprises a micro-fluidic chip and a detection frame, wherein the detection frame is connected with a clamp table for placing the chip, the micro-fluidic chip comprises a substrate sheet and a key valve body, a channel sheet is arranged on the upper side of the substrate sheet, a plurality of micro-fluidic branch channels are arranged at the downward end of the channel sheet, a plurality of liquid storage tanks which are in one-to-one correspondence with the micro-fluidic branch channels are arranged on the channel sheet, a plurality of liquid inlet holes which are in one-to-one correspondence with the liquid storage tanks are arranged on the channel sheet, the key valve body is respectively connected with the channel sheet and the substrate sheet, a fluid through hole is arranged on the key valve body, the fluid through hole can rotatably cover the area where the micro-fluidic, the other end of the fluid through hole is communicated with a circulation area, and a first microfluid main channel and a negative pressure interface are sequentially arranged on a channel sheet at the outward end of the circulation area; the invention has simple structure, simple operation and sensitive detection.
Description
Technical Field
The invention belongs to the technical field of medical treatment, and particularly relates to a micro-fluidic device for multi-target quantitative detection.
Background
In vitro diagnosis refers to a method of detecting a sample such as blood or body fluid of a human body, and diagnosing body functions or disease information. In vitro diagnosis products can rapidly and accurately diagnose diseases in early stage, provide effective technical support for disease prevention, prognosis evaluation and the like, and the development of highly automated, integrated and miniaturized diagnosis equipment becomes the main component of the in vitro diagnosis market.
Currently, most of the immunodiagnosis in hospitals and laboratories uses an elisa plate or a centrifuge tube as a reaction vessel and is provided with a large-scale detection instrument. Although the method has high stability and good repeatability, a large amount of reagents are required, the detection cost is high, the detection time is long, special technicians are often required for operation, and the method is lack of flexibility and portability. In some rural clinics or remote areas, the diagnosis of disease is difficult to perform without standard experimental conditions and professionals. The chip comprises a gas control channel layer, an organic molecular polymer film and a microfluid channel layer, wherein the organic molecular polymer film is directly clamped between the gas control channel layer and the microfluid channel layer, a pneumatic pump needs to be externally connected during use, the channel structure of the gas control layer is troublesome in design and difficult to process, and in addition, the chip has a small application range and is not beneficial to popularization of the microfluidic chip.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides the micro-fluidic device for multi-target quantitative detection and the detection method thereof, solves the technical problem of complex structure in the prior art, and has simple structure and high flexibility.
The purpose of the invention is realized as follows: a micro-fluidic device for multi-target quantitative detection comprises a micro-fluidic chip and a detection frame, wherein a clamp table is connected onto the detection frame, a positioning groove is formed in the clamp table, the micro-fluidic chip comprises a substrate sheet and a key valve body, a channel sheet is tightly attached to the upper side of the substrate sheet, an upper mounting hole is formed in the channel sheet, a lower mounting hole corresponding to the upper mounting hole is formed in the substrate sheet, a plurality of micro-fluidic branch channels respectively communicated with the upper mounting hole are arranged at the downward end of the channel sheet, a liquid storage tank is formed in the channel sheet at the end, far away from the upper mounting hole, of each micro-fluidic branch channel, a plurality of liquid inlet holes corresponding to the liquid storage tanks one by one are formed in the channel sheet, the liquid inlet holes are communicated with the corresponding liquid storage tanks, the key valve body sequentially penetrates through the upper mounting hole and the lower mounting hole to be respectively connected with the channel sheet and the substrate, the fluid through hole can rotatably cover the area where the microfluid branch channel is located, the lowest position of the fluid through hole is not lower than the height of the lower side of the circulation area, the downward end of the channel sheet is provided with the circulation area, a microfluid main channel I and a negative pressure interface, when one end of the fluid through hole is communicated with any microfluid branch channel, the other end of the fluid through hole is communicated with the circulation area, the first microfluid main channel is communicated with the circulation area, the negative pressure interface is arranged far away from the circulation area and communicated with the first microfluid main channel, the substrate sheet can be just arranged in the positioning groove, the detection frame at the front end and the rear end of the clamp table is connected with a lifting push rod, a negative pressure interface board is fixed on one downward side of the push rod, a connecting pipe corresponding to the negative pressure interface is arranged on the negative pressure interface board, an inner cavity of the connecting pipe is communicated with the negative pressure interface, and a peristaltic pump is fixedly connected to the detection frame at the outward end of the clamp table.
According to the invention, the peristaltic pump is a miniature negative pressure type peristaltic pump, the peristaltic pump is connected with the connecting pipe, a PDMS gasket which is tightly attached to the negative pressure interface is arranged on the lower side of the negative pressure interface board, the key valve body is in interference fit with the channel sheet, and the key valve body can rotate; injecting corresponding reagents into each liquid storage tank through the liquid inlet holes; the invention has simple structure, convenient operation, strong applicability, convenient carrying and high flexibility, and can realize the detection of various markers; can be applied to the work of detecting different markers.
As a further improvement of the invention, a flaring buffer channel is arranged on the channel sheet at one end of the microfluid branch channel far away from the liquid storage tank, one end of the flaring buffer channel far away from the microfluid branch channel can be communicated with the fluid through hole, the fluid through hole covers the area where the flaring buffer channel is located, and the sectional area of the flaring buffer channel is larger than that of the microfluid branch channel; in this design, the flow rate of reagent from the microfluidic branch into the fluidic via is prevented from being too fast and generating a large pressure.
In order to further make things convenient for the location of key valve body, still include the base, the base piece is connected at the base upside, the base top is connected with the mount pad, and it has the connecting hole to open on the mount pad, and the periphery on key valve body upper portion is equipped with the location arch, the key valve body passes the connecting hole in proper order, goes up the mounting hole and is connected with the base with lower mounting hole, and the protruding upside of contradicting at the mount pad in the location.
In order to further improve the accuracy of the experiment, a second microfluidic main channel and at least two branch reaction channels are formed on a channel sheet at one end of the first microfluidic main channel, which is far away from the circulation area, one ends of the plurality of branch reaction channels, which are far away from the main fluid channel, are converged together and communicated with one end of the second microfluidic main channel, which is far away from the branch reaction channels, is connected with a negative pressure interface, an antigen-antibody pattern sheet is arranged on the lower side of the channel sheet, and the antigen-antibody pattern sheet covers the area where the branch reaction channels are located; the antigen-antibody pattern piece has the capability of adsorbing antigens/antibodies, and on the basis, the pattern piece is further processed, so that the pattern piece has the capability of adsorbing various antigens or antibodies with different concentrations, and the dynamic range detection of various markers is realized.
As a further improvement of the invention, one upward end of the substrate sheet between the circulation area and the negative pressure interface is provided with a buffer groove, and the substrate sheet below the negative pressure interface is provided with a waste liquid groove.
As a further improvement of the invention, the buffer groove corresponds to the position of the second microfluidic main channel.
In order to rotate the key valve body conveniently, a rotating handle is arranged on the upper side of the key valve body.
In order to further realize the detection of the microfluidic chip, the front end and the rear end of the clamp table are respectively and fixedly connected with a first fixing plate and a second fixing plate, a linear driver is fixedly connected onto the first fixing plate, a push rod which extends downwards and can do reciprocating linear movement in the height direction is connected onto the linear driver, a driving motor is fixedly connected onto the second fixing plate, a driving shaft is connected onto the driving motor, a clamping jaw is connected onto one end, extending downwards, of the driving shaft, and a connecting groove which is used for just accommodating a rotating handle is formed in the clamping jaw; in the design, before detection, the linear driver acts, and the push rod descends to enable the gasket to be tightly attached to the upper side of the channel sheet; the clamping jaw is inserted on the rotating handle through the connecting groove, so that the driving of the rotating handle is realized, and the installation is convenient.
In order to further facilitate the installation of the clamp table, one end of the clamp table, which is far away from the peristaltic pump, is provided with a handle, the detection frame is provided with a sliding port, the clamp table is inserted into the detection frame along the sliding port, and a limiting step of the clamp table is abutted against the outer side of the detection frame.
Drawings
FIG. 1 is a front view of the detecting device of the present invention
FIG. 2 is a perspective view of the detecting unit of the present invention.
Fig. 3 is a perspective view of the jig stage according to the present invention.
Fig. 4 is a perspective view of the inspection frame of the present invention.
Fig. 5 is a perspective view of the microfluidic chip according to the present invention.
Fig. 6 is a three-dimensional structure view of the channel sheet of the present invention.
FIG. 7 is a perspective view of a substrate sheet according to the present invention.
Fig. 8 is an exploded view of a microfluidic chip according to the present invention.
FIG. 9 is a structural view of an antigen-antibody pattern sheet of the present invention on a channel sheet.
FIG. 10 is a diagram of the optimization of the flow rate simulation for the side-stream reaction channel in the present invention.
FIG. 11 is a velocity profile of a cross-section of two of three substream reaction channels.
FIG. 12 is a velocity profile of the cross-section of the middle of three substream reaction channels.
FIG. 13 is a linear graph of the detection of three inflammatory markers of C-reactive protein (CRP), Procalcitonin (PCT) and interleukin 6 (IL-6).
FIG. 14(1-4) is a dot-matrix diagram in which CRP capture antibodies with a concentration of 5. mu.g/mL were adsorbed on aluminum foil paper, nitrocellulose paper, a silica gel sheet, and a PDMS film, respectively, and exposed to light for 15s on a chemiluminescence apparatus after immunoreaction.
FIGS. 15(1-4) are specific dot-matrix diagrams for detecting chemiluminescence of three inflammatory markers, C-reactive protein (CRP), Procalcitonin (PCT), and interleukin 6 (IL-6).
The detection device comprises a detection frame 1, a sliding port 101, a clamp table 2, a handle 201, a limit step 202, a positioning groove 203, a first fixing plate 3, a peristaltic pump 4, a microfluidic chip 5, a substrate sheet 501, a base 502, a liquid inlet hole 503, a mounting seat 504, a positioning bulge 505, a rotating handle 506, a key valve body 507, a channel sheet 508, a negative pressure interface 509, a first microfluidic main channel 510, a reservoir 511, a microfluidic branch channel 512, a flaring buffer channel 513, a flow area 514, a branch reaction channel 515, a second microfluidic main channel 516, a lower mounting hole 517, a buffer groove 518, a waste liquid groove 519, an upper mounting hole 520, an antigen-antibody pattern sheet 521, a gasket 6, a push rod 7, a second fixing plate 8, a linear driver 9, a negative pressure interface board 10, a connecting pipe 11 and a driving motor 12.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Example 1
As shown in fig. 1 to 9, the microfluidic device for multi-target quantitative detection comprises a microfluidic chip 5 and a detection frame 1, wherein the detection frame 1 is connected with a fixture table 2, the fixture table 2 is provided with a positioning groove 203, the microfluidic chip 5 comprises a base 502, the upper side of the base 502 is connected with a substrate piece 501, the base 502 and the substrate piece 501 can be just placed in the positioning groove 203, the detection frame 1 at the front end and the rear end of the fixture table 2 is connected with a lifting push rod 7, the downward side of the push rod 7 is fixed with a negative pressure interface board 10, the upper side of the substrate piece 501 is tightly attached with a channel piece 508, the base 502 is connected with a mounting seat 504, the mounting seat 504 is abutted against the upper side of the channel piece 508, the channel piece 508 is provided with an upper mounting hole 520, the substrate piece is provided with a lower mounting hole 517 corresponding to the upper mounting hole 520, the mounting seat is provided with a connecting hole, the periphery of the upper part of a key, An upper mounting hole 520 and a lower mounting hole 517 are connected with the base 502, the positioning protrusion 505 is abutted against the upper side of the mounting seat 504, a plurality of micro-fluid branch passages 512 respectively communicated with the upper mounting hole 520 are arranged at the downward end of the passage sheet 508, in the embodiment, six micro-fluid branch passages 512 are arranged, a liquid storage tank 511 is arranged on the passage sheet 508 at one end of the micro-fluid branch passage 512 far away from the upper mounting hole 520, a plurality of liquid inlet holes 503 one-to-one corresponding to the liquid storage tank 511 are arranged on the passage sheet 508, a rotating handle 506 is arranged on the upper side of the key valve body 507, the key valve body 507 sequentially penetrates through the upper mounting hole 520 and the lower mounting hole 517 to be respectively connected with the passage sheet 508 and the base sheet 501, a fluid through hole 13 is arranged on the periphery of the key valve body 507, a buffer flaring passage 513 is arranged on the passage sheet 508 at one end of the micro-fluid branch passage 512 far away from the liquid storage, the fluid through hole 13 covers the area where the flaring buffer channel 513 is located, the sectional area of the flaring buffer channel 513 is larger than the sectional area of the microfluidic branch channel 512, the depth of the flaring buffer channel 513 is larger than the depth of the microfluidic branch channel 512, the lowest position of the fluid through hole 13 is not lower than the height of the lower side of the circulation area 514, in the embodiment, the lowest position of the fluid through hole 13 is flush with the lower side of the circulation area 514, the downward end of the channel sheet 508 is provided with the circulation area 514, the microfluidic main channel I510 and the negative pressure interface 509, when one end of the fluid through hole 13 is communicated with any microfluidic branch channel 512, the other end of the fluid through hole 13 is communicated with the circulation area 514, the microfluidic main channel I510 is communicated with the circulation area 514, the negative pressure interface 509 is arranged far away from the circulation area 514, the negative pressure interface 509 is communicated with the microfluidic main channel I, the inner cavity of the connecting pipe 11 is communicated with the negative pressure interface 509, the clamp table 2 is fixedly connected with the peristaltic pump 4 on the detection frame 1 at one end facing outwards, one end of the clamp table 2, which is far away from the peristaltic pump 4, is provided with a handle 201, the detection frame 1 is provided with a sliding port 101, the clamp table 2 is inserted into the detection frame 1 along the sliding port 101, and the limiting step 202 of the clamp table 2 is abutted to the outer side of the detection frame 1.
In order to further realize the detection of the microfluidic chip 5, the front end and the rear end of the fixture table 2 are respectively and fixedly connected with a first fixing plate 3 and a second fixing plate 8, the first fixing plate 3 is fixedly connected with a linear driver 9, the linear driver 9 is connected with a push rod 7 which extends downwards and can do reciprocating linear movement in the height direction, the second fixing plate 8 is fixedly connected with a driving motor 12, the driving motor 12 is connected with a driving shaft, one end of the driving shaft extending downwards is connected with a clamping jaw, and the clamping jaw is provided with a connecting groove which just accommodates the rotating handle 506.
In order to further improve the accuracy of the experiment, a second microfluidic main channel 516 and at least two branch reaction channels 515 are formed on the channel sheet 508 at one end of the first microfluidic main channel 510, which is far away from the flow-through area 514, in this embodiment, the number of the branch reaction channels 515 is preferably 3, one ends of the plurality of branch reaction channels 515, which are far away from the fluid main channel, are gathered together and communicated with one end of the second microfluidic main channel 516, which is far away from the branch reaction channels 515, is connected with the negative pressure interface 509, an antigen-antibody pattern sheet 521 is arranged on the lower side of the channel sheet 508, and the antigen-antibody pattern sheet 521 covers the area where the; the upward end of the substrate 501 between the flow area 514 and the negative pressure port 509 is provided with a buffer groove 518, the substrate 501 below the negative pressure port 509 is provided with a waste liquid groove 519, and the buffer groove 518 corresponds to the position of the second microfluidic main channel 516.
In the invention, the peristaltic pump 4 is a miniature negative pressure type peristaltic pump 4, the peristaltic pump 4 is connected with a connecting pipe 11, the lower side of a negative pressure interface board 10 is provided with a PDMS gasket 6 which is tightly attached to a negative pressure interface 509, and a key valve body 507 is in interference fit with a channel piece 508; as shown in FIG. 14(1-4), the coating efficiency of the aluminum foil paper (FIG. 14-1) and the nitrocellulose paper (FIG. 14-2) is poor, and the adsorption capacity of the silica gel sheet (FIG. 14-3) is equivalent to that of the PDMS film (FIG. 14-4); the PDMS film has higher permeability and good adsorption capacity, but has higher cost; the preparation method of the EVOH nanofiber film comprises the following steps of dissolving EVOH particles in DMAC (dimethylacetamide), and intensively stirring for 5 hours in a water bath kettle at 60 ℃ to prepare a transparent EVOH spinning solution with the concentration of 8 wt%; then, an electrostatic spinning machine is utilized to apply 25KV voltage, the voltage is sprayed on the silica gel sheet at a spraying distance of 20cm at a perfusion speed of 5mL/h, and the electrospinning time is 15 min; in addition, the circulation area 514 is semicircular, the peripheral diameter is 7-11 mm, and the height is 0.2-0.6 mm, in the embodiment, the peripheral diameter is preferably 9mm, and the height is preferably 0.4 mm; the width of the micro-fluid branch channel 512 is 0.1-0.3 mm, the height is 0.1-0.3 mm, the width is preferably 0.2mm, and the height is preferably 0.2 mm; the width of the flaring buffer channel 513 is 0.4-0.8 mm, and the height is 0.4-0.8 mm; the width of the flaring buffer channel 513 is preferably 0.4mm, and the height is preferably 0.4 mm; the width of the microfluid main channel is 0.4-0.8 mm, the height is 0.2-0.6 mm, the width of the microfluid main channel is preferably 0.6mm, and the height is preferably 0.4 mm; the bifurcation angle of two adjacent branch reaction channels 515 is 30-90 degrees, the length is 10-14 mm, the width is 0.3-0.7 mm, the height is 0.3-0.7 mm, the distance between two adjacent branch reaction channels 515 is 1.5-2.5 mm, preferably, the bifurcation angle is 60 degrees, the total length of the branch reaction channels 515 is 12mm, the width is 0.5mm, the height is 0.4mm, and the distance between the two adjacent branch reaction channels is 2 mm; the widths of the upper end and the lower end of the middle area of the branch reaction channel 515 are adjusted to be 0.3mm, and the length is 2 mm; as shown in fig. 10, the optimized velocity distribution of the three reaction branch channels is simulated by using multi-physics simulation software, and as shown in fig. 11 and 12, the velocities of the two side reaction branch channels and the middle reaction branch channel are respectively shown; under the condition that the flow rate of the first microfluid main channel 510 is 10mm/s, the maximum flow rate of the middle area of the branch reaction channel 515 is 5.8mm/s, and the maximum flow rate of the two side areas of the branch reaction channel 515 is 6.1mm/s, so that the reagents can basically and synchronously flow into different branch reaction channels 515 through the parameter setting of each channel, and uniform flow division is realized; when assembling the chip, removing dust on the surfaces of the PDMS channel layer 1 and the PDMS substrate layer 2, and punching holes in 6 liquid storage tanks 511 by using a 2mm puncher to form a liquid inlet hole 503; then, carrying out hydrophilization surface treatment by using a plasma cleaning machine, bombarding the surfaces of the PDMS channel piece 508 and the PDMS substrate piece 501 for 60 seconds by using plasma under the conditions of 200W power and 1.5L/min oxygen flow, and breaking silicon-oxygen bonds on the surfaces; placing an antigen-antibody pattern sheet 521 at three tributary reaction channels 515 of a channel sheet 508, aligning upper mounting holes 520 of the channel sheet 508 with lower mounting holes 517 on a substrate sheet 501, and extruding bubbles in the double PDMS layer by hand to make the bubbles tightly bonded; assembling the mounting seat 504 and the base 502 by using a PC rod pin, fixedly mounting a bonded double PDMS sheet at a gap formed in the middle, inserting the key valve body 507 into a mounting hole of the double PDMS sheet and a connecting hole of the mounting seat 504, and abutting the positioning bulge 505 against the upper side of the mounting seat 504, thereby completing the chip assembly of the invention; the invention has more compact structure, convenient installation and operation, can realize the detection of various markers, and has strong applicability, high flexibility and sensitive detection; can be applied to the work of detecting different markers.
In addition, it should be noted that,
the term "PDMS" refers to: polydimethylsiloxane.
The term "EVOH" means: ethylene-vinyl alcohol copolymers.
The term "DMAC" refers to: n, N-dimethylacetamide.
Example 2
The method of performing detection using the detection apparatus of embodiment 1, in which the number of the reservoirs 511 is 6, and the reservoirs 511 which are sequentially communicated with the fluid passage hole 13 in accordance with the rotation direction of the key valve body 507 are referred to as a first reservoir 511, a second reservoir 511, a third reservoir 511, a fourth reservoir 511, a fifth reservoir 511, and a sixth reservoir 511, includes the steps of,
(1) injecting 35 μ L of phosphate buffered saline (PBS, PH 7.4) containing 3% Bovine Serum Albumin (BSA) as a blocking agent into the first reservoir 511 at room temperature, injecting a standard solution of an inflammation marker into the second reservoir 511, and adding 35 μ L of PBS containing 0.05% V/V tween-20 as a washing solution into the third reservoir 511 and the fifth reservoir 511, respectively; injecting a detection antibody labeled by horseradish peroxidase into the fourth reservoir 511; injecting Luminol-H into the sixth reservoir 5112O2A chemiluminescent substrate;
(2) controlling the action of a driving motor 12, rotating a key valve body 507, stopping the action of the driving motor 12 when a fluid through hole 13 is communicated with a first liquid storage tank 511, controlling the work of a peristaltic pump 4, enabling a sealant to sequentially pass through a microfluid branch channel 512, a flaring buffer channel 513, the fluid through hole 13, a circulation zone 514 and a microfluid main channel 510 to enter a branch reaction channel 515 area, stopping the action of the peristaltic pump 4 when the sealant enters a buffer tank 518, stopping for 20 minutes, sealing the vacant sites of three capture antibody strips on an antigen-antibody pattern sheet 521, and after sealing for 20 minutes, pneumatically driving the peristaltic pump 4 to suck the sealant out to enter a waste liquid tank 519 or the buffer tank 518, and stopping the action of the peristaltic pump 4;
(3) the driving motor 12 continues to act, the key valve body 507 rotates, the fluid through hole 13 is communicated with the second liquid storage tank 511, the driving motor 12 stops acting, the peristaltic pump 4 works, a detection sample in the second liquid storage tank 511 sequentially passes through the microfluid branch channel 512, the flaring buffer channel 513, the fluid through hole 13, the circulation area 514 and the microfluid main channel 510 to enter the branch reaction channel 515 area to generate specific immunoreaction with a capture antibody, when the detection sample enters the waste liquid tank 519, the peristaltic pump 4 stops acting, after the static reaction is carried out for 20 minutes, the peristaltic pump 4 is pneumatically operated to suck out all standard solutions flowing into each channel from the second liquid storage tank 511, and then the standard solutions enter the waste liquid tank 519 or the buffer tank 518, and the peristaltic pump 4 stops acting;
(4) the driving motor 12 acts again, the key valve body 507 rotates to enable the fluid through hole 13 to be communicated with the third liquid storage tank 511, the driving motor 12 stops acting, the peristaltic pump 4 acts, the washing liquid in the third liquid storage tank 511 passes through each channel and is used for washing the channel and the antigen or antibody which does not generate immune reaction, and when the washing liquid is completely sucked into the buffer groove 518 or the waste liquid groove 519, the peristaltic pump 4 stops acting;
(5) the driving motor 12 continues to act, the key valve body 507 rotates, the fluid through hole 13 is communicated with the fourth liquid storage tank 511, the driving motor 12 stops acting, the peristaltic pump 4 acts, the detection antibodies in the fourth liquid storage tank 511 sequentially pass through all channels to enter a branch reaction channel 515 area, the detection antibodies and the detection samples generate specific variation reaction, when the detection antibodies enter the buffer tank 518, the peristaltic pump 4 stops acting, after standing reaction for 20 minutes, the peristaltic pump 4 is started, the detection antibodies flowing into all channels from the fourth liquid storage tank 511 are sucked out and enter the buffer tank 518 or a waste liquid tank 519, and the peristaltic pump 4 stops acting;
(6) the driving motor 12 operates again, the key valve body 507 rotates to enable the fluid through hole 13 to be communicated with the fifth liquid storage tank 511, the driving motor 12 stops operating, the washing liquid in the fifth liquid storage tank 511 passes through the whole channel and is used for washing the channel and the antigen or antibody which does not generate immune reaction, and when the buffer liquid is completely sucked into the buffer tank 518 or the waste liquid tank 519, the peristaltic pump 4 stops operating;
(7) the driving motor 12 continues to operate, the key valve body 507 rotates to enable the fluid through hole 13 to be communicated with the sixth liquid storage tank 511, the driving motor 12 stops operating, the peristaltic pump 4 operates, and Luminol-H2O2The chemiluminescent substrate sequentially passes through each channel and enters the branch reaction channel 515 area, chemical reaction occurs in the branch reaction channel 515 area, and the peristaltic pump 4 stops acting;
(8) after all the samples are added and the reaction is finished, a full-automatic chemiluminescence imager is used for shooting the chip, and the exposure time is 30 s;
the standard solution in the step (1) is a standard solution mixed by three inflammation markers, and comprises a CRP standard substance of 10 mu g/mL, a PCT standard substance of 5ng/mL and an IL-6 standard substance of 400pg/mL, wherein the volume ratio of the three standard substances is 1:1: 1; the fourth reservoir 511 is filled with 2.5. mu.g/mL horseradish peroxidase (HRP) -labeled CRP detection antibody, 4. mu.g/mLHRP-labeled PCT detection antibody, and 10. mu.g/mLHRP-labeled IL-6 detection antibody at a volume ratio of 1:1: 1.
Example 3
A linear detection method for three inflammation marker standard products by using a detection device, in particular,
the antigen-antibody pattern sheet 521 was coated with 15 μ g/mL of PCT capture antibody in advance, and the CRP standard was prepared at concentrations of 0 μ g/mL, 2 μ g/mL, 4 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 160 μ g/mL, and 320 μ g/mL using PBS buffer (PH 7.4) as a diluent, and 30 μ L of the CRP standard was added to the second reservoir 511; to the fourth reservoir 511 was added 30 μ L of a 2.5 μ g/mL HRP-labeled CRP antibody solution; the rest steps are the same as the example 2, each standard substance is respectively measured for 3 times by using 3 micro-fluidic chips 5, and a quantitative analysis system is used for fitting and calculating the average value of 9 points of each concentration according to the proportion between the CRP concentration and the chemiluminescence gray value and drawing a standard curve;
the antigen-antibody pattern sheet 521 is coated with 15 μ g/mL of PCT capture antibody in advance, and PCT standard substances are prepared at concentrations of 0ng/mL, 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 8ng/mL, 32ng/mL, 64ng/mL, and 128ng/mL using PBS buffer (PH 7.4) as a diluent, and 30 μ L of PCT standard substances are added to the second reservoir 511; to the fourth reservoir 511 was added 30 μ L of a 5 μ g/mL HRP-labeled PCT antibody solution; the other steps are the same as the two embodiments, each standard substance is respectively measured for 3 times by using 3 microfluidic chips 5, and a quantitative analysis system is used for fitting and calculating the average value of 9 points of each concentration according to the proportion between the PCT concentration and the chemiluminescence gray value, and drawing a standard curve;
an IL-6 capture antibody of 30 μ g/mL was coated on the antigen-antibody pattern sheet 521 of all EVOH nanofiber films in advance, and an IL-6 standard was prepared at concentrations of 0pg/mL, 20pg/mL, 40pg/mL, 80pg/mL, 160pg/mL, 320pg/mL, 640pg/mL, 1320pg/mL, and 2640pg/mL using PBS buffer (PH 7.4) as a diluent, and 30 μ L was taken and added to the second reservoir 511; to the fourth reservoir 511 was added 30. mu.L of a 10. mu.g/mL HRP-labeled IL-6 antibody solution; the other steps are the same as the two embodiments, each standard substance is respectively measured for 3 times by using 3 microfluidic chips 5, and a quantitative analysis system calculates the average value of 9 points of each concentration in a fitting manner according to the proportion between the IL-6 concentration and the chemiluminescence gray value, and draws a standard curve.
The quantitative analysis system comprises a computer and a full-automatic chemiluminescence apparatus comprising a freezing CCD camera, the freezing CCD camera is connected with the computer, the freezing CCD camera completes exposure shooting and transmits shot chemiluminescence signals to the computer, and analysis software in the computer analyzes images of the received chemiluminescence signals.
As shown in FIG. 11, the linear ranges of the three inflammation markers CRP, PCT and IL-6 are respectively 2-80 μ g/mL, 0.5-64 ng/mL and 40-1320 pg/mL, and the lowest detection lines are respectively 1.32 μ g/mL, 0.36ng/mL and 27.9pg/mL, so that the detection range is wide and multiple biomarkers can be dynamically detected.
The specificity of the three inflammation markers is verified, specifically,
adding a mixed standard sample of CRP and PCT to the second reservoir 511; adding a detection antibody corresponding to the standard substance into the fourth liquid storage tank 511, and performing the rest detection steps in the same manner as in example 2, wherein the exposure time of the chemiluminescence apparatus is 30s, so as to obtain an image shown in fig. 15-1;
adding a mixed standard sample of CRP and IL-6 to the second reservoir 511; the detection antibody corresponding to the standard was added to the fourth reservoir 511, and the rest of the detection procedure was the same as in example 2. The exposure time of the chemiluminescence apparatus was 30s, and the image of fig. 15-2 was obtained;
adding a mixed standard sample of PCT and IL-6 to the second reservoir 511; the detection antibody corresponding to the standard was added to the fourth reservoir 511, and the rest of the detection steps were the same as in example 2, and the exposure time of the chemiluminescence apparatus was 30 seconds, thereby obtaining images of fig. 15 to 3.
Analysis results show that the key valve core piece does not have a chemiluminescence signal of a third marker except for detecting two markers added with the standard, and the silicon rubber sheet and the EVOH fiber film are used as immunoreaction substrates and have good specificity.
Finally, CRP, PCT, IL-6 standards and their corresponding capture antibodies were added to the second reservoir 511 and the fourth reservoir 511, respectively, and the concentrations of the three markers were quantitatively determined, with the rest steps being the same as in example 2, and the exposure time of the chemiluminescence apparatus being 30s, to obtain FIGS. 15-4.
In addition, when the specificity experiment is verified, the concentration of the CRP standard substance is 20 mug/mL, the concentration of the PCT standard substance is 5ng/mL, the concentration of the IL-6 standard substance is 100pg/mL, and the volume ratio of the three standard substances is 1:1: 1; the concentration of the HRP-labeled CRP antibody solution is 2.5. mu.g/mL, the concentration of the HRP-labeled PCT antibody solution is 5. mu.g/mL, the concentration of the HRP-labeled IL-6 antibody solution is 10. mu.g/mL, and the volume ratio of the three antibody solutions is 1:1: 1.
The present invention is not limited to the above-mentioned embodiments, and based on the technical solutions disclosed in the present invention, those skilled in the art can make some substitutions and modifications to some technical features without creative efforts according to the disclosed technical contents, and these substitutions and modifications are all within the protection scope of the present invention.
Claims (9)
1. A multi-target quantitative detection microfluidic device is characterized in that: the key valve comprises a micro-fluidic chip and a detection frame, wherein a clamp table is connected to the detection frame, a positioning groove is formed in the clamp table, the micro-fluidic chip comprises a substrate sheet and a key valve body, a channel sheet is tightly attached to the upper side of the substrate sheet, an upper mounting hole is formed in the channel sheet, a lower mounting hole corresponding to the upper mounting hole is formed in the substrate sheet, a plurality of micro-fluidic branch channels respectively communicated with the upper mounting hole are arranged at the downward end of the channel sheet, a liquid storage tank is formed in the channel sheet at the end, far away from the upper mounting hole, of each micro-fluidic branch channel, a plurality of liquid inlet holes corresponding to the liquid storage tanks one by one are formed in the channel sheet, the liquid inlet holes are communicated with the corresponding liquid storage tanks, the key valve body sequentially penetrates through the upper mounting hole and the lower mounting hole to be respectively connected with the channel sheet and the substrate sheet, a fluid through hole is formed, the lowest position of the fluid through hole is not lower than the height of the lower side of the circulation area, the downward end of the channel piece is provided with a circulation area, a microfluid main channel I and a negative pressure interface, when one end of the fluid through hole is communicated with any microfluid branch channel, the other end of the fluid through hole is communicated with the circulation area, the microfluid main channel I is communicated with the circulation area, the negative pressure interface is far away from the circulation area, the negative pressure interface is communicated with the microfluid main channel I, the substrate piece can be just arranged in the positioning groove, the detection frame at the front end and the rear end of the clamp table is connected with a liftable push rod, the downward side of the push rod is fixed with a negative pressure interface board, the negative pressure interface board is provided with a connecting pipe corresponding to the position of the negative pressure interface, the inner cavity of the connecting.
2. The microfluidic device for multi-target quantitative detection according to claim 1, wherein a flaring buffer channel is formed on the channel piece at one end of the microfluidic branch channel away from the liquid storage tank, one end of the flaring buffer channel away from the microfluidic branch channel can be communicated with the fluid through hole, the fluid through hole covers the area where the flaring buffer channel is located in a rotating manner, and the sectional area of the flaring buffer channel is larger than that of the microfluidic branch channel.
3. The microfluidic device for quantitative detection of multiple targets according to claim 1, wherein the microfluidic chip further comprises a base, the base can be just placed in the positioning groove, the substrate sheet is connected to the upper side of the base, a mounting seat is connected to the upper side of the base, a connecting hole is formed in the mounting seat, a positioning protrusion is arranged on the periphery of the upper portion of the key valve body, the key valve body sequentially penetrates through the connecting hole, the upper mounting hole and the lower mounting hole to be connected with the base, and the positioning protrusion abuts against the upper side of the mounting seat.
4. The microfluidic device according to claim 1, wherein a second microfluidic main channel and at least two branch reaction channels are formed on the channel sheet at an end of the first microfluidic main channel away from the circulation area, ends of the branch reaction channels away from the main fluid channel are converged and communicated with one end of the second microfluidic main channel, one end of the second microfluidic main channel away from the branch reaction channels is connected with a negative pressure port, and an antigen-antibody pattern sheet is disposed on a lower side of the channel sheet and covers an area where the branch reaction channels are located.
5. The microfluidic device for multi-target quantitative detection according to any one of claims 1 to 4, wherein: the upward end of the substrate between the circulation area and the negative pressure interface is provided with a buffer groove, and the substrate below the negative pressure interface is provided with a waste liquid groove.
6. The microfluidic device for multi-target quantitative detection according to claim 5, wherein the buffer groove corresponds to the position of the second microfluidic main channel.
7. The microfluidic device for multi-target quantitative detection according to any one of claims 1 to 4, wherein a turning handle is arranged on the upper side of the key valve body.
8. The microfluidic device according to claim 7, wherein a first fixing plate and a second fixing plate are fixedly connected to the front end and the rear end of the fixture table, respectively, a linear actuator is fixedly connected to the first fixing plate, a push rod extending downward and capable of performing reciprocating linear movement in the height direction is connected to the linear actuator, a driving motor is fixedly connected to the second fixing plate, a driving shaft is connected to the driving motor, a clamping jaw is connected to one end of the driving shaft extending downward, and a connecting groove for accommodating a rotating handle is formed in the clamping jaw.
9. The microfluidic device according to any one of claims 1 to 4, wherein a handle is disposed at an end of the fixture table away from the peristaltic pump, the detection frame is provided with a sliding opening, the fixture table is inserted into the detection frame along the sliding opening, and a limiting step of the fixture table abuts against an outer side of the detection frame.
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