CN113769803B - Micro-fluidic chip for military detection of infection marker and detection method thereof - Google Patents
Micro-fluidic chip for military detection of infection marker and detection method thereof Download PDFInfo
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- CN113769803B CN113769803B CN202111158238.3A CN202111158238A CN113769803B CN 113769803 B CN113769803 B CN 113769803B CN 202111158238 A CN202111158238 A CN 202111158238A CN 113769803 B CN113769803 B CN 113769803B
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Abstract
The invention discloses a micro-fluidic chip for military detection of infection markers and a detection method thereof, and the micro-fluidic chip comprises a connecting component, wherein the connecting component comprises an upper valve frame, the lower side of the upper valve frame is connected with a lower valve frame, the front end and the rear end of the upper valve frame are connected with the lower valve frame through a first short pin and a second short pin, a mounting port is arranged between the upper valve frame and the lower valve frame, the upper valve frame and the lower valve frame are respectively provided with an upper connecting hole and a lower connecting hole which are coaxial, a micro-fluidic reaction component for detection of the infection markers is connected between the upper valve frame and the lower valve frame, the micro-fluidic component and the connecting component are connected with a control valve which can move in the height direction, and all solutions in the micro-fluidic component are injected according to a detection sequence through the control valve; the invention has simple structure, convenient detection and short detection time.
Description
Technical Field
The invention relates to the technical field of infection marker detection, in particular to a micro-fluidic chip for military detection of infection markers and a detection method thereof.
Background
The point-of-care testing (POCT) can provide a detection result nearby and rapidly and provide an instructive basis for diagnosis and treatment. Compared with the traditional detection method, the POCT equipment has the characteristics of convenience in carrying, easiness in operation and high detection speed. For common military diseases, the POCT equipment can provide a more convenient detection mode, and can effectively ensure the life health and safety of soldiers in training fields, battlefields and other complex military environments.
Infections can range from minor skin trauma to severe organ failure, which is very common in military training, battlefield, and other settings. Severe infections may lead to sepsis, which increases the risk of death by 6-10% per 1 hour delay. There are also a number of classifications of infection, with bacterial and viral infections being common and different treatments for different infections. Therefore, the early and rapid diagnosis of infection is of great utility and importance in the military.
Microfluidics is the research direction of multiple disciplines such as integrated machinery, materials, biology, chemistry. The micro-fluidic chip can integrate a complete biochemical analysis process in a smaller volume, has the characteristics of high reaction speed, low reagent consumption, high flux, low cost and high automation degree, and is an important research direction for the development of instant examination. Enzyme-linked immunosorbent assay is a mature medical detection method, but the steps are complicated, the specialization degree is high, and a large-scale analytical instrument is relied on. Reagent premixing through a micromixer can simplify the steps of the ELISA method, and greatly shorten the detection time. The chemiluminescence method uses chemiluminescence as a marker, and after the reaction of a labeled enzyme and a substrate, the chemiluminescence method can emit light quickly, and signals can be read conveniently, so that the chemiluminescence method is a mature detection means in a microfluidic chip. Therefore, the micro-fluidic chip is taken as a platform to develop military POCT equipment, and the early rapid diagnosis of the infection marker has reliable technical basis.
In the micro-fluidic chip in the prior art, the movement steps of fluid among the multi-layer chips are complex, only the traditional ELISA method is integrated, the detection time is not reduced, in addition, the whole volume of the chip is large, the operation steps are complex, the specialization degree of the operation is high, the chip is difficult to be used as POCT equipment, and the chip is difficult to be applied to the fields of complex environments such as military and the like.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the prior art infection marker detection.
Therefore, the invention aims to provide a micro-fluidic chip for military detection of infection markers and a detection method thereof, which have the advantages of convenient detection, high detection efficiency and realization of combined detection of multiple markers in a wide concentration range.
In order to solve the technical problems, the invention provides the following technical scheme: a micro-fluidic chip for military detection of infection markers and a detection method thereof, which comprises,
the connecting assembly comprises an upper valve frame, a lower valve frame is connected to the lower side of the upper valve frame, a mounting opening is formed between the upper valve frame and the lower valve frame, and an upper connecting hole and a lower connecting hole which are coaxial are formed in the upper valve frame and the lower valve frame respectively;
the micro-fluidic reaction assembly is connected between the upper valve frame and the lower valve frame and comprises an upper chip body and a lower chip body which are connected together from top to bottom, an upper valve hole and a lower valve hole are formed in the upper chip body and the lower chip body respectively, a mixed micro-channel is arranged at the downward end of the upper chip body, a plurality of liquid storage tanks are distributed in the mixed micro-channel, a detection micro-channel is arranged at the downward end of the upper chip body, which is far away from one end of the control valve, of the mixed micro-channel, a waste liquid micro-channel is arranged on the upper chip body, which is far away from one end of the mixed micro-channel, a negative pressure interface is formed in the upper chip body, which is far away from one end of the mixed micro-channel, of the waste liquid micro-channel, a waste liquid tank is arranged at the upward end of the lower chip body, the waste liquid micro-channel is communicated with the waste liquid tank, and a detection layer is connected between the upper chip body and the lower chip body at the detection micro-channel;
the control valve can be pressed down and sequentially penetrates through the upper connecting hole, the upper valve hole, the lower valve hole and the lower connecting hole, and the control valve can enable different liquid storage tanks to be communicated with the mixing micro-channel.
As a preferable scheme of the micro-fluidic chip for military detection of the infection marker, the invention comprises the following steps: the one end of liquid reserve tank is sealed, be equipped with inlet channel on the last chip body of the liquid reserve tank other end, inlet channel keeps away from the one end and the last valve hole intercommunication of liquid reserve tank, is equipped with a plurality of and the control channel of liquid reserve tank one-to-one that set up at the interval on the direction of height on the control valve, control channel's one end and the liquid reserve tank intercommunication that corresponds, all control channel's the other end homoenergetic and the head end intercommunication of mixing the microchannel, it can cover control channel's the other end to mix the microchannel.
As a preferable scheme of the micro-fluidic chip for military detection of the infection marker, the invention comprises the following steps: a plurality of positive pressure interfaces which are in one-to-one correspondence with the liquid storage tanks are distributed on the upper chip body, and the positive pressure interfaces are communicated with the corresponding liquid storage tanks.
As a preferable scheme of the micro-fluidic chip for military detection of the infection marker, the invention comprises the following steps: the detection layer is coated with a plurality of bands for capturing antibodies, the bands for capturing antibodies are arranged in a straight line and in parallel, the length of the bands for capturing antibodies is 10mm, the width of the bands for capturing antibodies is 400 mu m, and the interval is 3mm.
As a preferable scheme of the micro-fluidic chip for military detection of infection markers, the invention comprises the following steps: the mixed micro-channel is formed by connecting a plurality of semi-elliptical micro-channels end to end, the major axis of each semi-ellipse is 5mm, the minor axis is 3mm, the width is 400 +/-5 mu m, and the depth is 400 +/-8 mu m.
As a preferable scheme of the micro-fluidic chip for military detection of infection markers, the invention comprises the following steps: the waste liquid micro-channel is a 17mm long linear micro-channel.
As a preferable scheme of the micro-fluidic chip for military detection of infection markers, the invention comprises the following steps: be equipped with two down play liquid microchannel on the control valve, two down go out the liquid microchannel on the same height and respectively with two inlet channel one-to-ones that are close to, be equipped with at least one on the control valve of play liquid microchannel top down and go out the liquid microchannel, go up the one end homoenergetic of going out liquid microchannel and going out liquid microchannel down and the inlet channel intercommunication that corresponds, go up the other end homoenergetic of liquid microchannel and going out liquid microchannel down and mix the microchannel intercommunication.
A method for detection using a microfluidic chip for military detection of infection markers, comprising the steps of,
adding a standard solution of the antigen or a diluted serum sample into the first liquid storage pool;
adding CRP and PCT detection antibodies coupled with HRP, IL-6 detection antibodies coupled with B and HRP coupled with SA into a second liquid storage pool;
adding PBST cleaning solution into the third liquid storage tank;
adding a chemiluminescent substrate to the fourth reservoir;
a lower pressure control valve is used for enabling the two lower liquid outlet micro-channels to be respectively communicated with the first liquid storage tank and the second liquid storage tank, pumping the solution in the first liquid storage tank and the solution in the second liquid storage tank into the mixed micro-channel, enabling the antigen and the detection antibody to be uniformly mixed, enabling the mixed solution to enter the detection micro-channel, standing and incubating, and enabling the capture antibody coated on the detection layer to specifically adsorb the antigen;
pumping the reacted solution into a waste liquid pool;
a lower pressure control valve is used for communicating the lowest upper liquid outlet microchannel with the third liquid storage tank, the PBST cleaning solution in the third liquid storage tank is pumped into the mixed microchannel, and impurities which do not participate in the reaction are flushed into the waste liquid tank;
continuing to press a control valve, communicating the upper liquid outlet microchannel with the fourth liquid storage tank, pumping the chemiluminescent substrate in the fourth liquid storage tank into the detection microchannel, reacting hydrogen peroxide in the chemiluminescent substrate with HRP to generate oxygen free radicals, and oxidizing luminol in the chemiluminescent substrate by the oxygen free radicals to generate chemiluminescence;
the chip is placed in a chemiluminescence detection instrument for exposure, a transverse linear strip of the detection micro-channel and an antibody strip coated on the detection layer form a luminous lattice, chemiluminescence values of all points are obtained through measurement, and the average gray value of all points is subtracted from a background value to be used for calculating the concentration of a sample.
The invention has the beneficial effects that: the specified reagent is gated through the control valve, the sample and the detection antibody are uniformly mixed through the mixing micro-channel, the mixing effect is good, then the sample and the detection antibody react with the pre-coated capture antibody strip in the detection area, the substrate is added for reaction after the washing, the chemiluminescence is generated, the conversion is converted into the concentration of the marker, the detection time is short, and the operation is simple; the negative pressure interface can be used as a port for connecting a negative pressure pump during negative pressure driving, and can be used for balancing the internal and external pressures of the chip during positive pressure driving and fluid movement; the detection chip and the detection method are combined to detect the low-concentration IL-6, so that the difficulty of the combined detection is overcome, and the combined detection of the infection markers is realized; the method is particularly suitable for detection of complex environments such as military infection marker detection and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
fig. 1 is a three-dimensional structure diagram of a microfluidic chip according to the present invention.
Fig. 2 is an exploded view of the microfluidic chip according to the present invention.
Fig. 3 is a perspective view of the upper chip body according to the present invention.
Fig. 4 is a perspective view of a control valve according to the present invention.
Fig. 5 is a physical diagram of the prepared microfluidic chip.
FIG. 6 is a representation of the micro-channel of the microfluidic chip of the present invention.
Fig. 7 is a simulation result diagram of the mixing effect of the mixing channel in the microfluidic chip.
FIG. 8 is a diagram illustrating the detection principle of the detection method of the present invention.
FIG. 9 is a schematic view of the capture antibody coating of the detection layer of the present invention.
FIG. 10 is a graph showing the detection range of three infection markers for CRP, PCT and IL-6 in the present invention.
FIG. 11 is a linear graph of three infection markers for detecting CRP, PCT, and IL-6 in the present invention.
FIG. 12 is a luminescence dot-matrix diagram of the combined detection of three infection markers CRP, PCT and IL-6 in the present invention.
In the figure, 100 microfluidic reaction components, 101 lower chip body, 101a waste liquid pool, 101b lower valve hole, 102 upper chip body, 102a first positive pressure interface, 102b second positive pressure interface, 102c fourth positive pressure interface, 102d upper valve hole, 102e third positive pressure interface, 102f first liquid pool, 102g second liquid pool, 102h third liquid pool, 102i fourth liquid pool, 102j waste liquid microchannel, 102k detection microchannel, 102l mixing microchannel, 102m liquid inlet channel, 103 detection layer, 200 connection component, 201 lower valve frame, 201a lower connection hole, 202 upper valve frame, 202a connection hole, 203 first short pin, 204 second short pin, 300 control valve, 301 lower liquid outlet microchannel, 302 upper liquid outlet microchannel, 303 indication key.
Detailed Description
Before describing particular embodiments of the present invention, the terms used herein are defined as follows:
the term "PDMS" refers to: polydimethylsiloxane;
the term "CRP" refers to: c-reactive protein;
the term "PCT" refers to: procalcitonin;
the term "IL-6" refers to: interleukin-6;
the term "SA" means: streptavidin;
the term "B" means: biotin;
the term "HRP" means: horseradish peroxidase;
the term "BSA" refers to: bovine serum albumin;
the term "PBST" refers to: phosphate buffer containing 0.05% tween 20.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
Referring to fig. 1 to 4, a first embodiment of the present invention provides a microfluidic chip for military detection of infection markers, which has a simple structure and facilitates the joint detection of multiple markers.
A micro-fluidic chip for military detection of infection markers comprises a connecting assembly 200, wherein the connecting assembly 200 comprises an upper valve frame 202, a lower valve frame 201 is connected to the lower side of the upper valve frame 202, the front end and the rear end of the upper valve frame 202 are connected with the lower valve frame 201 through a first short pin 203 and a second short pin 204, a mounting port is formed between the upper valve frame 202 and the lower valve frame 201, an upper connecting hole 202a and a lower connecting hole 201a which are coaxial are formed in the upper valve frame 202 and the lower valve frame 201 respectively, a micro-fluidic reaction assembly 100 for detection of the infection markers is connected between the upper valve frame 202 and the lower valve frame 201, a control valve 300 capable of moving in the height direction is connected to the micro-fluidic assembly and the connecting assembly 200, and all solutions in the micro-fluidic assembly are injected according to a detection sequence through the control valve 300.
Further, the microfluidic reaction assembly 100 is connected between the upper valve frame 202 and the lower valve frame 201, the microfluidic reaction assembly 100 includes an upper chip body 102 and a lower chip body 101 which are connected together from top to bottom, an upper valve hole 102d and a lower valve hole 101b are respectively formed on the upper chip body 102 and the lower chip body 101, a mixed micro-channel 102l is arranged at the downward end of the upper chip body 102 and a plurality of liquid storage tanks are arranged, a detection micro-channel 102k is arranged at the downward end of the upper chip body 102, which is far away from one end of the control valve 300, of the mixed micro-channel 102l, a waste liquid micro-channel 102j is arranged on the upper chip body 102, which is far away from one end of the mixed micro-channel 102l, of the waste liquid micro-channel 102j is provided with a negative pressure interface on the upper chip body 102, which is far away from one end of the mixed micro-channel 102l, a waste liquid pool 101a is arranged at the upward end of the lower chip body 101, the waste liquid micro-channel 102j is communicated with the waste liquid pool 101a, and a detection layer 103 is connected between the upper chip body 102 and the lower chip body 101 at the position of the detection micro-channel 102 k.
Further, the control valve 300 can be pressed down to sequentially pass through the upper connecting hole 202a, the upper valve hole 102d, the lower valve hole 101b and the lower connecting hole 201a, the control valve 300 is in interference fit with the upper chip body 102 and the lower chip body 101, and the control valve 300 can enable different liquid storage pools to be communicated with the mixing micro-channel 102 l.
Further, one end of the liquid storage tank is closed, liquid inlet channels 102m are arranged on the upper chip body 102 at the other end of the liquid storage tank, one end, far away from the liquid storage tank, of each liquid inlet channel 102m is communicated with an upper valve hole 102d, a plurality of control channels which are arranged at intervals in the height direction and correspond to the liquid storage tanks one to one are arranged on the control valve 300, one ends of the control channels are communicated with the corresponding liquid storage tanks, the other ends of all the control channels can be communicated with the head ends of the mixing micro-channels 102l, and the mixing micro-channels 102l can cover the other ends of the control channels; a plurality of positive pressure interfaces which are in one-to-one correspondence with the liquid storage tanks are arranged on the upper chip body 102, and the positive pressure interfaces are communicated with the corresponding liquid storage tanks.
Furthermore, the detection layer 103 is coated with a plurality of bands for capturing antibodies, the bands for capturing antibodies are arranged linearly and in parallel, the length of the bands for capturing antibodies is 10mm, the width of the bands for capturing antibodies is 400 μm, the intervals are 3mm, and the size of the detection layer 103 is 10mm, 8mm and 0.1mm.
Furthermore, the optimal shape and size parameters of the mixed micro-channel 102l are obtained through a large number of experiments, the mixed micro-channel 102l is formed by connecting a plurality of semi-elliptical micro-channels end to end, the major axis of each semi-ellipse is 5mm, the minor axis is 3mm, the width is 400 +/-5 mu m, and the depth is 400 +/-8 mu m; the waste liquid micro-channel 102j is a linear micro-channel with the length of 17 mm; the mixing efficiency of the fluid in the mixing microchannel 102L is simulated by using CIMSOL software, two solutions with the concentrations of 0mol/L and 1mol/L are arranged at an inlet, the mixing effect is simulated under the condition that the reynolds numbers are 0.4, 4 and 40 respectively, if the two solutions with different concentrations can be mixed into a solution with uniform concentration, the mixing microchannel 102L has good mixing capability, as shown in fig. 7, the mixing efficiency of the mixing microchannel 102L is over 0.995 for different reynolds numbers, which indicates that the mixing microchannel 102L can complete high-efficiency mixing under any reynolds number no matter the mixing microchannel is driven by an automatic instrument or manually controlled, and the mixing efficiency is a precondition that the microfluidic chip in the application is suitable for complex environments such as military conditions.
Further, two lower liquid outlet micro-channels 301 are arranged on the control valve 300, the two lower liquid outlet micro-channels 301 are at the same height and respectively correspond to the two adjacent liquid inlet channels 102m one by one, at least one upper liquid outlet micro-channel 302 is arranged on the control valve 300 above the lower liquid outlet micro-channels 301, one end of each of the upper liquid outlet micro-channels 302 and one end of each of the lower liquid outlet micro-channels 301 can be communicated with the corresponding liquid inlet channel 102m, and the other end of each of the upper liquid outlet micro-channels 302 and the other end of each of the lower liquid outlet micro-channels 301 can be communicated with the mixing micro-channel 102 l; the solution is communicated with a designated liquid storage tank selectively through a lower pressure control valve 300, so that the solutions are sequentially mixed and reacted according to a set sequence; an indication key 303 is provided on the outer side of the upper portion of the control valve 300, and when the control valve 300 is installed, the indication key 303 corresponds to the position of one end of the mixing micro-channel 102 l.
The upper chip body 102 and the lower chip body 101 are made of PDMS, and can be manufactured by adopting methods such as photoetching, one-time injection molding, mold manufacturing and the like, wherein the methods are mature methods in the industry; the detection layer 103 is made of silica gel; in this embodiment, four liquid reservoirs are provided, the four liquid reservoirs are respectively a first liquid reservoir 102f, a second liquid reservoir 102g, a third liquid reservoir 102h and a fourth liquid reservoir 102i, two lower liquid outlet channels can be respectively communicated with the first liquid reservoir 102f and the second liquid reservoir 102g, a lower upper liquid outlet channel can be communicated with the third liquid reservoir 102h, an upper liquid outlet channel can be communicated with the fourth liquid reservoir 102i, and the four positive pressure interfaces are respectively a first positive pressure interface 102a communicated with the first liquid reservoir 102f, a second positive pressure interface 102b communicated with the second liquid reservoir 102g, a third positive pressure interface 102e communicated with the third liquid reservoir 102h and a fourth positive pressure interface 102c communicated with the fourth liquid reservoir 102 i.
Example 2
A second embodiment of the present invention differs from the first embodiment in that it provides a method for detection using a microfluidic chip for military detection of infection markers, comprising the steps of:
adding a standard solution of antigen or a diluted serum sample to the first reservoir 102 f;
adding CRP, PCT detection antibody conjugated with HRP, IL-6 detection antibody conjugated with B, HRP conjugated with SA to the second reservoir 102 g;
adding PBST wash solution to the third reservoir 102 h;
adding a chemiluminescent substrate to the fourth reservoir 102 i;
a lower pressure control valve 300, which is used for communicating the two lower liquid outlet microchannels 301 with the first liquid storage tank 102f and the second liquid storage tank 102g respectively, pumping the solution in the first liquid storage tank and the solution in the second liquid storage tank into a mixing microchannel 102l, uniformly mixing the antigen and the detection antibody, allowing the mixed solution to enter a detection microchannel 102k, standing for incubation, and specifically adsorbing the antigen by the capture antibody coated on the detection layer 103 to form a sandwich structure of the capture antibody-antigen-detection antibody;
pumping the reacted solution into a waste liquid pool 101a;
a lower pressure control valve 300, which makes the lowest upper outlet liquid microchannel 302 communicate with the third liquid storage tank, and pumps the PBST cleaning solution in the third liquid storage tank into the mixing microchannel 102l, and flushes the impurities which do not participate in the reaction into the waste liquid tank 101a;
continuing to control the lower pressure valve 300 to communicate the upper outlet liquid microchannel 302 with the fourth liquid reservoir, pumping the chemiluminescent substrate in the fourth liquid reservoir into the detection microchannel 102k, reacting hydrogen peroxide in the chemiluminescent substrate with HRP to generate oxygen radicals, and oxidizing luminol in the chemiluminescent substrate by the oxygen radicals to generate chemiluminescence;
the chip is put into a chemiluminescence detection instrument for exposure, a luminous lattice is formed by a horizontal linear strip of the detection microchannel 102k and an antibody strip coated on the detection layer 103, chemiluminescence values of all points are obtained by measurement, and the average gray value of the chemiluminescence values is subtracted from a background value to be used for calculating the concentration of a sample.
Example 3
A third embodiment of the present invention is different from the 1 st and 2 nd embodiments in that it provides a method of assembling a microfluidic chip, which includes the steps of:
cleaning an upper chip body 102 and a lower chip body 101, placing the cleaned upper chip body and the cleaned lower chip body in a plasma cleaning machine, cutting off a surface silicon-oxygen key, placing a detection layer 103 at a specified position between the upper chip body and the lower chip body, and pressing tightly, wherein a micro-channel is not required to be pressed in the process; after the upper chip body 102 and the lower chip body 101 are tightly bonded, the lower valve frame 201 is placed on the lower side of the lower chip body 101, and the first short pin 203 and the second short pin 204 are inserted to realize the connection of the upper valve frame 202 and the lower valve frame 201; finally, the control valve 300 is inserted, the index key 303 being aligned at the inlet of the mixing microchannel 102 l; the pictures and the real object images of all the parts are shown in figure 5, and figure 6 is a size representation diagram of all the parts in the chip, and the manufacturing error is extremely small and is within 2 percent.
Example 4
A fourth embodiment of the present invention is different from any one of embodiments 1 to 3 in that it provides a method for pretreating a microfluidic chip, comprising the steps of:
injecting capture antibody solutions of CRP, PCT and IL-6 into the channels respectively, washing the micro-channels with PBST washing liquor after 20min, and removing capture antibodies which are not successfully coated, wherein the capture antibodies are coated on the detection layer 103;
after the chip is assembled, 50 μ l of 5% BSA solution is added into any liquid storage tank, the corresponding liquid outlet micro-channel on the control valve 300 is pressed down to the height of the corresponding liquid storage tank, the BSA solution is pumped into the micro-channel until the whole mixing micro-channel 102l and the detection micro-channel 102k are filled, the incubation is carried out for 15 minutes in a static way so as to seal the micro-channel and the unsealed vacant sites on the detection layer 103 and avoid non-specific adsorption, finally, the BSA solution is completely pumped into the waste liquid tank 101a, the control valve 300 is restored to the original position, and the pretreatment of the microfluidic chip is completed.
Example 5
The fifth embodiment of the present invention is different from any of embodiments 1 to 4 in that it provides a method for jointly detecting CRP, PCT, and IL-6, comprising the steps of:
the detection layer 1032 is coated with one capture antibody of CRP, PCT and IL-6 respectively, and the concentrations of the capture antibodies of three infection markers of CRP, PCT and IL-6 are respectively 40 mug/mL, 60 mug/mL and 80 mug/mL;
after assembling and pretreating the chip, 30 μ L of the standard sample, 10 μ L each of CRP, PCT, IL-6, was added to the first reservoir 102 f;
adding 40 μ L of CRP conjugated with HRP, PCT detection antibody, IL-6 conjugated with B, and HRP conjugated with SA to 102g of the second liquid storage pool, wherein the final concentration of each of the four solutions is 10 μ L, and the final concentration is 25 μ g/mL, 50 μ g/mL, and 4 μ g/mL; add 50. Mu.L of PBST wash solution to the third reservoir 102h, add 35. Mu.L of chemiluminescent substrate to the fourth reservoir 102 i; completing the loading of each reagent;
a hose is connected with a negative pressure peristaltic pump or an injector at the negative pressure interface, and fluid in the chip can be driven by using negative pressure;
connecting a syringe pump or an injector at the first positive pressure interface 102a, the second positive pressure interface 102b, the third positive pressure interface 102e and the fourth positive pressure interface 102c by using hoses, wherein positive pressure can be used for driving fluid in the chip;
the control valve 300 is pressed to the height that the lower liquid outlet micro-channel 301 is aligned with the liquid inlet channel 102m, so that the solutions in the first liquid storage tank 102f and the second liquid storage tank 102g are fully mixed in the mixed micro-channel 102l, flow into the detection micro-channel 102k after reaction, and are statically incubated for 15 minutes, so that the mixed solution is subjected to specific adsorption in the detection micro-channel 102k to form a double-antibody sandwich structure;
then, all liquid in the micro-channel is sucked into a waste liquid pool 101a;
the lower pressure control valve 300 is arranged to the height of the lower upper liquid outlet micro-channel 302 aligned with the liquid inlet channel 102m, so that PBST washing liquid in the third liquid storage tank 102h enters the micro-channel, continuous washing for 15 seconds is completed until all PBST washing liquid enters the waste liquid tank 101a, and impurities which do not participate in the reaction are flushed into the waste liquid tank 101a;
continuing to press the lower pressure control valve 300 to the level where the upper outlet microchannel 302 is aligned with the inlet channel 102m, so that the chemiluminescent substrate flows into the detection microchannel 102k, reacts with the HRP, and then flows into the waste liquid tank 101a;
after the reaction process is finished, putting the chip into a chemiluminescence detector for exposure for 20s;
three linear channels of the detection micro-channel 102k are crossed with three capture antibody bands on the silica gel plate, each marker of CRP, PCT and IL-6 has three detection points, nine detection points are formed by symbiosis, images are processed to obtain chemiluminescence values of all points, and the chemiluminescence values can be used for calculating the concentration of a sample by subtracting background values from the average gray value of the points.
CRP was detected in the ranges of 0.16. Mu.g/mL, 0.31. Mu.g/mL, 0.63. Mu.g/mL, 1.25. Mu.g/mL, 2.5. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL and 80. Mu.g/mL, respectively, and the results are shown in FIG. 10; as shown in FIG. 11, the CRP obtained by fitting the points was in a linear relationship of 1.25. Mu.g/mL, 2.5. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, or 40. Mu.g/mL.
PCT in the ranges of 0.1ng/mL, 0.2ng/mL, 0.4ng/mL, 0.8ng/mL, 1.6ng/mL, 3.2ng/mL, 6.4ng/mL, 12.8ng/mL, 25.6ng/mL and 51.2ng/mL are detected respectively, and the result is shown in FIG. 10; by fitting each point, PCT showed good linearity in the ranges of 0.4ng/mL, 0.8ng/mL, 1.6ng/mL, 3.2ng/mL, 6.4ng/mL and 12.8ng/mL, as shown in FIG. 11.
IL-6 was detected in the ranges of 12.5pg/mL, 25pg/mL, 50pg/mL, 100pg/mL, 200pg/mL, 400pg/mL, 800pg/mL, 1.6ng/mL, 3.2ng/mL, and 6.4ng/mL, respectively, and the results are shown in FIG. 10; as shown in FIG. 11, the linear relationship of IL-6 was found to be good in the ranges of 50pg/mL, 100pg/mL, 200pg/mL, 400pg/mL, 800pg/mL, and 1.6 ng/mL.
From the above, the micro-fluidic chip and the method of the invention are used to realize triple detection of infection markers in a wide concentration range, and a common chemiluminescence method is adopted for CRP and PCT with high concentration in serum, a plurality of B are coupled on a detection antibody, a plurality of HRP are coupled on SA, B can be combined with a plurality of SA, so that more HRP can be combined on the detection antibody, and IL-6 with low concentration can generate strong chemiluminescence signals, so that signal amplification is carried out, and the infection markers in a wide concentration range can be jointly detected in one chip; the luminescent dot matrix for joint detection of three infection markers CRP, PCT and IL-6 is shown in figure 12, each marker generates three bright spots in one detection, the bright spots can be accurately read in the linear range of the concentration of the marker, and the signal intensity between the bright spots of the same marker is uniform and the repeatability is good. .
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (6)
1. The method for detecting by using the micro-fluidic chip for military detection of the infection marker is characterized by comprising the following steps: the micro-fluidic chip comprises a connecting assembly (200), the connecting assembly (200) comprises an upper valve frame (202), a lower valve frame (201) is connected to the lower side of the upper valve frame (202), and an upper connecting hole (202 a) and a lower connecting hole (201 a) which are coaxial are formed in the upper valve frame (202) and the lower valve frame (201) respectively;
the micro-fluidic reaction assembly (100) is connected between an upper valve frame (202) and a lower valve frame (201), the micro-fluidic reaction assembly (100) comprises an upper chip body (102) and a lower chip body (101) which are connected together from top to bottom, the upper chip body (102) and the lower chip body (101) are respectively provided with an upper valve hole (102 d) and a lower valve hole (101 b), one downward end of the upper chip body (102) is provided with a mixed micro-channel (102 l) and a plurality of liquid storage tanks are distributed, one downward end of the upper chip body (102) far away from one end of the control valve (300) of the mixed micro-channel (102 l) is provided with a detection micro-channel (102 k), the detection device is characterized in that a waste liquid micro-channel (102 j) is arranged on an upper chip body (102) at one end, far away from a mixed micro-channel (102 l), of the detection micro-channel (102 k), a negative pressure interface is arranged on the upper chip body (102) at one end, far away from the mixed micro-channel (102 l), of the waste liquid micro-channel (102 j), a waste liquid pool (101 a) is arranged at one upward end of the lower chip body (101), the waste liquid micro-channel (102 j) is communicated with the waste liquid pool (101 a), a detection layer (103) is connected between the upper chip body (102) and the lower chip body (101) at the position of the detection micro-channel (102 k), and a plurality of strips for capturing antibodies wrap the detection layer (103), the capture antibody strips are in a straight line and are arranged in parallel;
the control valve (300) can be pressed downwards to sequentially penetrate through the upper connecting hole (202 a), the upper valve hole (102 d), the lower valve hole (101 b) and the lower connecting hole (201 a), two lower liquid outlet micro channels (301) are arranged on the control valve (300), the two lower liquid outlet micro channels (301) are arranged at the same height and respectively correspond to the two liquid inlet channels (102 m) which are close to each other one by one, at least one upper liquid outlet micro channel (302) is arranged on the control valve (300) above the lower liquid outlet micro channels (301), one ends of the upper liquid outlet micro channel (302) and one end of the lower liquid outlet micro channel (301) can be communicated with the corresponding liquid inlet channels (102 m), and the other ends of the upper liquid outlet micro channel (302) and the lower liquid outlet micro channel (301) can be communicated with the mixing micro channel (102 l);
which comprises the following steps of (a) preparing,
adding a standard solution of the antigen or a diluted serum sample into the first liquid storage pool;
adding CRP and PCT detection antibodies coupled with HRP, IL-6 detection antibodies coupled with B and HRP coupled with SA into a second liquid storage pool;
adding PBST cleaning solution into the third liquid storage tank;
adding a chemiluminescent substrate to the fourth reservoir;
a lower pressure control valve (300) is used for enabling the two lower liquid outlet micro-channels (301) to be respectively communicated with the first liquid storage tank and the second liquid storage tank, the solution in the first liquid storage tank and the solution in the second liquid storage tank are pumped into a mixing micro-channel (102 l), so that the antigen and the detection antibody are uniformly mixed, the mixed solution enters a detection micro-channel (102 k), standing incubation is carried out, and the capture antibody coated on the detection layer (103) specifically adsorbs the antigen;
pumping the reacted solution into a waste liquid pool (101 a);
a lower pressure control valve (300) which enables the lowest upper liquid outlet microchannel (302) to be communicated with the third liquid storage tank, the PBST cleaning solution in the third liquid storage tank is pumped into the mixing microchannel (102 l), and impurities which do not participate in the reaction are flushed into the waste liquid tank (101 a);
continuing to press the control valve (300) downwards, enabling the upper liquid outlet micro-channel (302) to be communicated with the fourth liquid storage tank, pumping the chemiluminescent substrate in the fourth liquid storage tank into the detection micro-channel (102 k), enabling hydrogen peroxide in the chemiluminescent substrate to react with HRP to generate oxygen radicals, and enabling the oxygen radicals to oxidize luminol in the chemiluminescent substrate to generate chemiluminescence;
the chip is placed in a chemiluminescence detection instrument for exposure, a luminous lattice is formed by a transverse linear strip of the detection micro-channel (102 k) and an antibody strip coated on the detection layer (103), chemiluminescence values of all points are obtained through measurement, and the average gray value of all points is subtracted from a background value to be used for calculating the concentration of a sample.
2. The method of claim 1, wherein the detection is performed using a microfluidic chip for military detection of infection markers, wherein: the one end of liquid reserve tank is sealed, be equipped with inlet channel (102 m) on last chip body (102) of the liquid reserve tank other end, the one end and last valve hole (102 d) intercommunication of liquid reserve tank are kept away from in inlet channel (102 m), are equipped with a plurality of and the control channel of liquid reserve tank one-to-one that set up at the interval on height direction on control valve (300), control channel's one end and the liquid reserve tank intercommunication that corresponds, all control channel's the other end homoenergetic and the head end intercommunication that mixes microchannel (102 l), mix microchannel (102 l) and can cover control channel's the other end.
3. The method of claim 1 or 2, wherein the detection is performed using a microfluidic chip for military detection of infection markers, wherein: a plurality of positive pressure interfaces which correspond to the liquid storage tanks one by one are distributed on the upper chip body (102), and the positive pressure interfaces are communicated with the corresponding liquid storage tanks.
4. The method of claim 1 or 2, wherein the detection is performed using a microfluidic chip for military detection of infection markers, wherein: the capture antibody bands were 10mm in length and 400 μm in width, 3mm apart.
5. The method of claim 4, wherein the detection is performed using a microfluidic chip for military detection of infection markers, wherein: the mixed micro-channel (102 l) is formed by connecting a plurality of semi-elliptical micro-channels end to end, the major axis of each semi-ellipse is 5mm, the minor axis is 3mm, the width is 400 +/-5 mu m, and the depth is 400 +/-8 mu m.
6. The method of claim 5, wherein the detection is performed using a microfluidic chip for military detection of infection markers, wherein: the waste liquid micro-channel (102 j) is a linear micro-channel with the length of 17 mm.
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