CN113567421B - Quantitative detection method for multiple markers - Google Patents

Abstract

The invention discloses a multi-marker quantitative detection method, which includes the following steps: simultaneously extracting detection samples and detection antibodies into the mixing channel of the detection chip, and circulating the detection samples and detection antibodies in the mixing channel to fully mix. After the mixing is completed, the mixing The solution enters the reaction channel; it is incubated statically, and the capture antibody coated on the reaction layer corresponding to the position of the reaction channel specifically adsorbs the antigen, forming a double-antibody sandwich structure of capture antibody-antigen-detection antibody; the reacted solution is pumped into Waste liquid pool; the washing liquid flushes the impurities that did not participate in the reaction of the mixing channel and reaction channel and the unbound detection antibody into the waste liquid pool; the chemiluminescent substrate enters the reaction channel through the mixing channel, and reacts with the enzyme labeled on the detection antibody to produce chemical Luminescence; the invention can realize simultaneous sampling of multiple solutions and has high detection efficiency.

Description

A multi-marker quantitative detection method

Technical field

The invention relates to the technical field of marker detection, in particular to a multi-marker quantitative detection method.

Background technique

Point-of-care testing refers to a testing method that uses portable analytical instruments and supporting reagents to quickly obtain test results at the sampling site. Compared with traditional laboratory equipment, POCT equipment has many advantages such as portability, fast detection, low technical requirements for personnel operation, and easy maintenance. Especially for acute diseases, using POCT technology to conduct early self-screening in ambulances, homes, and communities is a powerful means to protect health.

Enzyme-linked immunosorbent assay (ELISA) is an immune reaction method based on the specific binding of antigens and antibodies. Based on ELISA, various methods such as the double-antibody sandwich method suitable for macromolecular proteins and the competition method suitable for small molecule proteins have been developed. In the existing technology, during detection, samples need to be added step by step and washed multiple times. There are problems such as long detection time and high reagent consumption, which places high technical requirements on operators.

Contents of the invention

The purpose of this section is to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section, the abstract and the title of the invention to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions cannot be used to limit the scope of the invention.

In view of the above and/or problems existing in existing marker detection, the present invention is proposed.

Therefore, the object of the present invention is to provide a method for quantitative detection of multiple markers, which achieves simultaneous sample injection, short detection time, low reagent consumption, and high detection efficiency.

In order to solve the above technical problems, the present invention provides the following technical solution: a multi-marker quantitative detection method, which includes the following steps:

At the same time, the detection sample and detection antibody are extracted into the mixing channel of the detection chip. The detection sample and detection antibody are circulated and pumped in the mixing channel to fully mix. After the mixing is completed, the mixed solution enters the reaction channel;

After static incubation, the capture antibody coated on the reaction layer corresponding to the position of the reaction channel specifically adsorbs the antigen, forming a double-antibody sandwich structure of capture antibody-antigen-detection antibody;

Pump the reacted solution into the waste pool;

The washing liquid flushes the impurities that did not participate in the reaction in the mixing channel and reaction channel and the unbound detection antibodies into the waste liquid pool;

The chemiluminescent substrate enters the reaction channel through the mixing channel and reacts with the detection antibody to produce chemiluminescence.

As a preferred solution of the multi-marker quantitative detection method of the present invention, the detection chip includes a chip assembly, and the chip assembly includes a first chip body and a second chip body on the lower side of the first chip body, The downward end of the first chip body is provided with several liquid reservoirs, a mixing channel and a reaction channel. The first chip body at one end of the reaction channel away from the liquid reservoir is provided with a first negative pressure interface. There are a number of sampling vents on the chip body that correspond to and are connected to the liquid storage tank. The liquid storage tank can be connected to one end of the mixing channel, and the other end of the mixing channel is connected to one end of the reaction channel. The reaction channel has The other end is connected to the waste liquid pool, the waste liquid pool is connected to the first negative pressure interface, and a reaction layer corresponding to the position of the reaction channel is provided between the first chip body and the second chip body.

As a preferred solution of the multi-marker quantitative detection method of the present invention, the first chip body is provided with an upper valve hole that allows the liquid reservoir to communicate with one end of the mixing channel.

As a preferred solution of the multi-marker quantitative detection method of the present invention, the detection chip further includes a control component, and the control component includes a mechanical valve, a first support bracket and a second support bracket connected together, The chip assembly is supported between the first support bracket and the second support bracket. The second chip body is provided with a lower valve hole that is coaxial with the upper valve hole. The first support bracket and the second support bracket are respectively provided with first guide hole and second guide hole. After the mechanical valve passes through the first guide hole and is pressed into the upper valve hole, the lower end of the mechanical valve can pass through the lower valve hole and the second guide hole in sequence.

As a preferred solution of the multi-marker quantitative detection method of the present invention, the first chip body is also provided with a second negative pressure interface, and the second negative pressure interface is connected to one end of the mixing channel.

As a preferred solution of the multi-marker quantitative detection method of the present invention, the mechanical valve is provided with a number of liquid outlet channels spaced in the height direction, and the liquid storage tank can pass through the corresponding liquid outlet channels. It is connected to one end of the mixing channel. When the liquid reservoir is just connected to the mixing channel through the liquid outlet channel, the liquid reservoir covers one end of the liquid outlet channel, and one end of the mixing channel covers the other end of the liquid outlet channel.

As a preferred solution of the multi-marker quantitative detection method of the present invention, the mechanical valve is provided with a guide protrusion. When the guide protrusion is aligned with one end of the mixing channel, the bottom liquid outlet channel is The liquid inlet end can correspond to multiple liquid reservoirs. The bottom liquid outlet channel has at least two liquid inlet ends. When the lower end of the bottom liquid outlet channel is flush with the lower side of the first chip body, there are at least two liquid storage tanks. The pools cover different liquid inlet ends respectively.

As a preferred solution of the multi-marker quantitative detection method of the present invention, before detection, align the guide protrusion with the position of one end of the mixing channel, press down the mechanical valve to press it into the upper valve hole, when the bottom When the liquid outlet channel is pressed to the lower side of the first chip body, the liquid reservoir corresponds to the two liquid inlet ends of the liquid outlet channel, stop pressing the mechanical valve, and transfer the detection sample, detection antibody, washing solution and chemiluminescent substrate. The materials are respectively injected into different liquid reservoirs, and the first negative pressure pump and the second negative pressure pump are respectively connected to the first negative pressure interface and the second negative pressure interface.

As a preferred solution of the multi-marker quantitative detection method of the present invention, when mixing the detection sample and the detection antibody, the first negative pressure pump operates first to pump the detection sample and the detection antibody into the mixing channel. When the reagent approaches the other end of the mixing channel, the first negative pressure pump stops and the second negative pressure pump works to flow the mixed solution flowing to the other end of the mixing channel in the direction of one end of the mixing channel to complete a cycle of mixing and repeat the above steps. , when the number of circulation mixing reaches the set threshold, the second negative pressure pump stops operating, and the first negative pressure pump sucks the mixed solution into the reaction channel for reaction.

As a preferred solution of the multi-marker quantitative detection method of the present invention, after the reaction is completed, the mechanical valve is pressed downward to make the middle liquid outlet channel correspond to the liquid storage tank in which the washing liquid is injected, and the first negative pressure The pump operates, and the washing liquid flows through the outlet channel, mixing channel and reaction channel in sequence and then rushes into the waste liquid pool to wash away the impurities and unbound detection antibodies on the reaction layer that do not participate in the reaction; continue to press the mechanical valve downward to allow the final The upper outlet channel corresponds to the liquid reservoir in which the chemiluminescent substrate is injected. The first negative pressure pump operates to pump the chemiluminescent substrate into the reaction channel, where it reacts with the enzyme labeled on the detection antibody to produce chemiluminescence; reaction The horizontal linear microchannels on the channel and the vertically coated antibody strips on the reaction layer form a luminescent lattice, and the chemiluminescence value of each point is measured.

Beneficial effects of the present invention: the use of the present invention can realize joint and synchronous detection of multiple markers, and can realize synchronous sampling. When different reagents are mixed, the reagents can be fully mixed evenly through multiple circulation mixing to avoid mixing in the mixing channel. Blockage, improve detection accuracy, no need for multiple washings, less reagent loss, and low detection cost.

Description of the drawings

In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings needed to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. Those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting any creative effort. in:

Figure 1 is a three-dimensional structural view of the present invention.

Figure 2 is an exploded structural diagram of the present invention.

Figure 3 is a three-dimensional structural view of the first chip body in the present invention.

Figure 4 shows the flow rate in the mixing channel and the reaction channel after the solution is mixed in the present invention.

Figure 5 is a diagram showing concentration changes in the mixing channel and the reaction channel during the solution mixing process in the present invention.

Figure 6 is a graph showing concentration changes in the mixing channel and the reaction channel after the solution is mixed in the present invention.

Figure 7 is an explanatory diagram of the detection principle of the present invention.

Figure 8 is a schematic diagram of the reaction layer capturing antibody coating in the present invention.

Figure 9 is a graph showing the detection range of three myocardial markers, cTnI, CK-MB, and Myo, according to the present invention.

Figure 10 is a linear graph for detecting three myocardial markers, cTnI, CK-MB, and Myo, in the present invention.

Figure 11 is a luminescence dot matrix diagram of the present invention for separate detection of three myocardial markers: cTnI, CK-MB, and Myo.

Figure 12 is a luminescence dot matrix diagram of the present invention for joint detection of three myocardial markers, cTnI, CK-MB, and Myo.

In the figure, 100 control component, 101 second support bracket, 101a second connection hole, 101b second guide hole, 102 first support bracket, 102a first connection hole, 102b first guide hole, 103 mechanical valve, 103a liquid outlet Channel, 103b guide protrusion, 104 connecting pin, 200 chip assembly, 201 first chip body, 201a injection vent, 201b upper valve hole, 201c first negative pressure interface, 201d second negative pressure interface, 201e liquid reservoir , 201e-1 first liquid reservoir, 201e-2 second liquid reservoir, 201e-3 third liquid reservoir, 201e-4 fourth liquid reservoir, 201f mixing channel, 201g reaction channel, 201g-1 connection micro Channel, 201g-2 linear microchannel, 201h waste liquid pool, 202 second chip body, 202a lower valve hole, 203 reaction layer.

Implementation

Before elaborating the technical solutions of the present invention, the terms used in this article are defined as follows:

The term "PC" refers to: polycarbonate;

The term "PDMS" refers to: polydimethylsiloxane;

The term "AMI" means: acute myocardial infarction;

The term "cTnI" refers to: cardiac troponin I;

The term "Myo" refers to: myoglobin;

The term "CK-MB" refers to: creatine kinase isoenzyme;

The term "HRP" refers to: horseradish peroxidase;

The term "BSA" refers to: bovine serum albumin;

The term "PBST" refers to: phosphate buffered saline containing 0.05% Tween 20.

In order to make the above objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention will be described in detail below with reference to the accompanying drawings.

Many specific details are set forth in the following description to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described here. Those skilled in the art can do so without departing from the connotation of the present invention. Similar generalizations are made, and therefore the present invention is not limited to the specific embodiments disclosed below.

Second, reference herein to "one embodiment" or "an embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. "In one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

Example

Refer to Figure 8, which is a first embodiment of the present invention. This embodiment is used to illustrate the coating of capture antibodies and the preprocessing of the detection chip, including the following steps:

Place a PDMS microfluidic chip with multiple linear channels vertically on the reaction layer 203. The channels are 500 μm wide, 300 μm deep, and arranged in parallel with a spacing of 3 mm. Inject the capture antibody solutions into the channels respectively, and according to the number of detection markers Different channels are selected for injection, and after 1 hour, the reaction layer 203 is washed three times with PBST washing solution to remove the capture antibodies that have not been successfully coated, and the capture antibodies are coated on the reaction layer 203.

After the detection chip is assembled, add 50 μl of 5% BSA solution into the first liquid reservoir 201e, press the mechanical valve 103 to make the bottom liquid outlet channel 103a correspond to the height of the first liquid reservoir 201e, and add the BSA solution Pump into the liquid outlet channel 103a until the entire channel is filled, and incubate statically for 15 minutes to seal the unblocked empty sites on the liquid outlet channel 103a and the reaction layer 203 to avoid non-specific adsorption. Finally, pump all the BSA solution into the detection chip. In the waste liquid pool 201h, the mechanical valve 103 is restored to its original position. At this point, the preprocessing of the chip is completed.

Example

The second embodiment of the present invention provides a multi-marker quantitative detection method with high detection efficiency, low consumables, and reduced detection costs.

A multi-marker quantitative detection method, including the following steps:

At the same time, the detection sample and the detection antibody are extracted into the mixing channel 201f of the detection chip. The detection sample and the detection antibody are circulated and pumped in the mixing channel 201f to mix thoroughly. After the mixing is completed, the mixed solution enters the reaction channel 201g;

After static incubation, the capture antibody coated on the reaction layer 203 corresponding to the position of the reaction channel 201g specifically adsorbs the antigen, and the antigen specifically combines with the detection antibody to form a double-antibody sandwich structure of capture antibody-antigen-detection antibody;

Pump the reacted solution into the waste liquid pool for 201 hours;

The washing liquid flushes the impurities that do not participate in the reaction and the unbound detection antibodies in the mixing channel 201f and the reaction channel 201g into the waste liquid pool 201h;

The chemiluminescent substrate enters the reaction channel 201g through the mixing channel 201f, and reacts with the detection antibody to produce chemiluminescence;

Put the chip assembly 200 into a chemiluminescence detection instrument and expose it. The horizontal linear microchannel 201g-2 of the reaction channel 201g and the vertically coated antibody strips on the reaction layer 203 form a luminescent lattice, and the chemiluminescence value of each point is measured. , their average gray value minus the background value can be used to calculate the sample concentration.

Among them, the detection sample is a diluted serum sample or a standard solution of the antigen, the detection antibody is a detection antibody coupled to HRP, the washing liquid is PBST washing liquid, and the chemiluminescent substrate is Luminol-H ₂ O ₂ chemiluminescent substrate.

Example

Referring to Figures 1 to 3, a third embodiment of the present invention is shown. The difference from the first embodiment is that the specific structure of the detection chip is described, and when the detection chip is used in Embodiment 2, the implementation Supplementary instructions for steps related to Example 2.

The detection chip includes a chip assembly 200. The chip assembly 200 includes a first chip body 201 and a second chip body 202 on the lower side of the first chip body 201. The downward end of the first chip body 201 is provided with several liquid reservoirs 201e, a Mixing channel 201f and a reaction channel 201g. The reaction channel 201g includes several linear microchannels 201g-2 that are parallel to each other. Two adjacent linear microchannels 201g-2 are connected together by an arc-shaped connecting microchannel 201g-1. , the linear microchannel 201g-2 at the first end is connected to the other end of the mixing channel 201f through the connecting microchannel 201g-1, and the first negative pressure interface 201c is provided on the first chip body 201 at the end of the reaction channel 201g away from the liquid reservoir 201e. , the first negative pressure interface 201c has a through hole with a diameter of 1 mm, which penetrates the first chip body 201. The first chip body 201 is provided with a number of sampling vents 201a that correspond to and are connected to the liquid reservoir 201e. The liquid pool 201e is composed of a rectangle with a length of 4 mm and a width of 3 mm and two semicircles with a diameter of 3 mm in the width direction. The depth of the liquid pool 201e is 3 mm and the total volume is about 60 μL. The liquid pool 201e can be mixed with One end of the channel 201f is connected. The first chip body 201 is provided with an upper valve hole 201b that enables the liquid reservoir 201e to communicate with one end of the mixing channel 201f. The other end of the mixing channel 201f is connected to one end of the reaction channel 201g. The mixing channel 201f One end gradually wraps around from the outside to the inside and then wraps around in the opposite direction from the inside out to the other end of the mixing channel 201f. The radius gradually decreases as it wraps from the outside to the inside. Specifically, the first half of the mixing channel 201f is formed by The center is composed of semicircles with radii of 7 mm, 5 mm, 3 mm and 1 mm connected end to end. The radius gradually becomes larger when going out from the innermost end. The second half of the mixing channel 201f is composed of the mixing channel 201f. The center is composed of semicircles with radii of 1 mm, 3 mm, 5 mm and 7 mm connected end to end. The other end of the reaction channel 201g is connected to the waste liquid pool 201h. The width of the reaction channel 201g is 400 μm and the depth is 400 μm. The distance between two adjacent linear microchannels 201g-2 is 3 mm. The length of the linear microchannel 201g-2 is 10 mm. The connecting microchannel 201g-1 is a semicircle with a diameter of 3 mm. The waste liquid pool 201h is connected to the first The negative pressure interface 201c is connected, and a reaction layer 203 corresponding to the location of the reaction channel 201g is provided between the first chip body 201 and the second chip body 202. The reaction layer 203 is silica gel with a length of 10 mm, a width of 10 mm, and a thickness of 0.1 mm. plate, the waste liquid pool 201h is composed of a rectangle with a length of 20 mm, a width of 5 mm, and two semicircles with a diameter of 5 mm in the width direction. The depth of the waste liquid pool 201h is 4 mm, and the total volume is about 400 μL. The waste liquid The pool 201h is used to accommodate the waste liquid during the detection process to prevent the waste liquid from overflowing and contaminating the environment and instruments. Its volume can carry all the reagents in the liquid storage pool 201e.

Further, the detection chip also includes a control component 100. The control component 100 includes a mechanical valve 103, a first support bracket 102 and a second support bracket 101 connected together. The chip component 200 is supported on the first support bracket 102 and the second support bracket. 101, the first support bracket 102 has first connection holes 102a at both ends, and the second support bracket has second connection holes 101a at both ends. The connection pin 104 is used to pass through the first connection hole 102a and the second connection hole 101a. The connecting hole 101a connects the first support bracket 102 and the second support bracket 101 together. The second chip body 202 has a lower valve hole 202a coaxial with the upper valve hole 201b. The first support bracket 102 and the second support bracket 101a are connected together. The bracket 101 has a first guide hole 102b and a second guide hole 101b respectively. After the mechanical valve 103 passes through the first guide hole 102b and is pressed into the upper valve hole 201b, the lower end of the mechanical valve 103 can pass through the lower valve hole in turn. 202a and the second guide hole 101b.

Furthermore, the first chip body 201 is also provided with a second negative pressure interface 201d. The second negative pressure interface 201d has the same structure as the first negative pressure interface 201c. The second negative pressure interface 201d is connected to one end of the mixing channel 201f. , the mechanical valve 103 is provided with a plurality of liquid outlet channels 103a spaced apart in the height direction. The liquid reservoir 201e can be connected to one end of the mixing channel 201f through the corresponding liquid outlet channel 103a. When the liquid reservoir 201e passes through the liquid outlet channel 103a When it is just connected to the mixing channel 201f, the liquid reservoir 201e covers one end of the liquid outlet channel 103a, and one end of the mixing channel 201f covers the other end of the liquid outlet channel 103a.

Furthermore, the mechanical valve 103 is provided with a guide protrusion 103b. When the guide protrusion 103b is aligned with one end of the mixing channel 201f, the liquid inlet end of the bottom liquid outlet channel 103a can correspond to multiple liquid storage pools 201e. The liquid outlet channel 103a has at least two liquid inlets. When the lower end of the lowermost liquid outlet channel 103a is flush with the lower side of the first chip body 201, there are at least two liquid reservoirs 201e covering different liquid inlets respectively; by The position setting of the guide protrusion 103b and one end of the mixing channel 201f realizes the circumferential positioning of the mechanical valve 103 and the chip assembly 200, which facilitates installation.

Before using the detection chip for detection, align the guide protrusion 103b with the position of one end of the mixing channel 201f, press down the mechanical valve 103 to press it into the upper valve hole 201b, when the bottom liquid outlet channel 103a is pressed to the first chip body 201 When the lower side is in the position, the two liquid storage pools 201e containing the detection sample and the detection antibody respectively correspond to the two liquid inlet ends of the liquid outlet channel 103a. Stop pressing the mechanical valve 103 to transfer the detection sample, detection antibody, and washing liquid. and the chemiluminescence substrate are respectively injected into different liquid reservoirs 201e, and the first negative pressure pump and the second negative pressure pump are respectively connected to the first negative pressure interface 201c and the second negative pressure interface 201d.

When using the detection chip to mix the detection sample and detection antibody, the first negative pressure pump operates first to pump the detection sample and detection antibody into the mixing channel 201f. When the pumped reagent approaches the other end of the mixing channel 201f, the first negative pressure pump Stop the action, and the second negative pressure pump works to flow the mixed solution flowing to the other end of the mixing channel 201f in the direction of one end of the mixing channel 201f to complete one cycle of mixing; repeat the above steps, when the number of cycle mixing times reaches the set threshold , in this embodiment, the number threshold is preferably 2 times. The second negative pressure pump stops operating, and the first negative pressure pump sucks the mixed solution into the reaction channel 201g for reaction. Referring to Figures 5 and 6, it can be seen that after continuous After cyclic mixing, the solution concentration is consistent, achieving uniform mixing of different samples.

After the reaction is completed, the mechanical valve 103 is pressed downward to make the middle liquid outlet channel 103a correspond to the liquid storage tank 201e in which the washing liquid is injected. The first negative pressure pump operates and the washing liquid passes through the liquid outlet channel 103a and the mixing channel 201f in sequence. and the reaction channel 201g and then rush into the waste liquid pool 201h to wash away the impurities and unbound detection antibodies on the reaction layer 203 that did not participate in the reaction; continue to press the mechanical valve 103 downward to make the uppermost liquid outlet channel 103a and the injected chemical Corresponding to the liquid reservoir 201e of the luminescent substrate, the first negative pressure pump operates to pump the chemiluminescent substrate into the reaction channel 201g, where it reacts with the enzyme labeled on the detection antibody to produce chemiluminescence; the linear microchannel on the reaction channel 201g 201g-2 and the vertically coated antibody strips on the reaction layer 203 form a luminescent dot matrix, and the chemiluminescence value of each point is measured.

Referring to Figure 7, (A) the preprocessing of the chip includes coating the capture antibody on the reactive layer 203, using BSA to seal the empty spots in the channel and loading reagents; (B) the antigen and detection antibody are mixed and reacted in the mixing channel 201f; (C) A specific immune reaction occurs in the reaction channel 201g, and the capture antibody specifically binds to the antigen; (D) Unreacted reagents are washed; (E) A chemiluminescent substrate is added to generate a chemiluminescent signal.

In the detection method, the detection chip is used to detect markers to achieve synchronous sample addition. During synchronous sample addition, the mixed solution after synchronous addition is mixed evenly by circulating the mixed solution to improve the detection accuracy; refer to Figure 4, Multi-physics simulation software was used to simulate the flow velocity of the fluid movement in the mixing channel 201f and the reaction channel 201g. The results showed that the fluid moved uniformly in the channel at a speed of 10 mm/s, and there was no fluid blockage, flow velocity mutation, or fluid distribution. The uneven situation indicates that the fluid movement in the chip is stable.

Referring to Figure 6, under the conditions of the continuous circulation pumping method in the mixing channel 201f and the specific structure of the mixing channel 201f, multi-physics simulation software was used to simulate the mixing effect of the fluid in the present invention. The results show that two fluids with different concentrations After passing through the mixing channel 201f, the fluid is mixed into a uniform concentration, indicating that the double helix microchannels 1-4 have a good mixing effect.

The use of the present invention saves the operation of one incubation and washing, can complete the quantitative detection of multiple markers within 17 minutes, and improves the detection efficiency; realizes the simultaneous injection and mixing of multiple reagents, and realizes through the combination of the chip component 200 and the detection method. The mixed solution is fully and evenly mixed, and the mixed solution has a uniform flow rate in each channel, enabling detection at multiple concentrations, and the detection is more reliable; it is suitable for the detection of most markers and has a wide range of applications.

Example

This is the fourth embodiment of the present invention. The difference between this embodiment and Examples 1 to 3 lies in the method of separately detecting three myocardial markers: cTnI, CK-MB, and Myo, which includes the following steps ,

Coat the reaction layer 203 with three cTnI capture antibodies. After assembling and preprocessing the chip, add 30 μL cTnI standard sample to the first reservoir 201e-1, and add 30 μL cTnI to the second reservoir 201e-2. For detection antibodies coupled to HRP, add 50 μL PBST washing solution to the third reservoir 201e-3, and add 35 μL Luminol-H ₂ O ₂ chemiluminescent substrate to the fourth reservoir 201e-4. Reagent loading is completed;

Connect the negative pressure pump with a hose at the first negative pressure interface 201c and the second negative pressure interface 201d for driving the fluid in the chip;

The mechanical valve 103 is pressed down so that the lowermost liquid outlet channel 103a corresponds to the height of the liquid reservoir 201e. The first negative pressure pump operates to move the liquid in the first liquid reservoir 201e-1 and the second liquid reservoir 201e-2. The reagent is pumped into the mixing channel 201f. After the set time is reached, the first negative pressure pump stops and the second negative pressure pump operates to pump the mixed reagent from the other end of the mixing channel 201f to the direction of one end of the mixing channel 201f. The set time is reached. After a certain period of time, the second negative pressure pump stops operating, and the above operation is repeatedly cycled until the number of cycles reaches the set cycle number threshold, and the second negative pressure pump completely stops operating to obtain a uniformly mixed sample;

The first negative pressure pump operates to pump the evenly mixed sample into the reaction channel 201g. The first negative pressure pump stops operating and incubates statically for 15 minutes, allowing the mixture to specifically adsorb in the reaction channel 201g to form a double-antibody sandwich structure. ;

Afterwards, the first negative pressure pump continues to operate, sucking all the liquid in the reaction channel 201g into the waste liquid pool 201h;

Press down the mechanical valve 103 to make the middle liquid outlet channel 103a correspond to the position of the liquid reservoir 201e. The first negative pressure pump operates to allow the PBST washing liquid in the third liquid reservoir 201e-3 to enter the channel. This takes about 15 seconds. Continuous washing until all the PBST washing buffer enters the waste liquid pool for 201h, impurities not participating in the reaction and unbound HRP-coupled detection antibodies can be flushed into the waste liquid pool for 201h;

Continue to press the mechanical valve 103 to make the top liquid outlet channel 103a correspond to the position of the liquid reservoir 201e. The first negative pressure pump operates to cause the Luminol-H ₂ O ₂ chemiluminescent substrate to flow into the reaction channel 201g and connect with the detection antibody. HRP reaction, and then flowed into the waste liquid pool for 201h;

After the reaction process is completed, put the chip into the chemiluminescence detector and expose it for 20 s;

Among them, the two liquid storage pools 201e connected to the lowermost liquid outlet channel 103a are respectively named the first liquid storage pool 201e-1 and the second liquid storage pool 201e-2, and the liquid storage pool 201e connected to the middle liquid outlet channel 103a The pool 201e is the third liquid storage pool 201e-3, and the liquid storage pool 201e connected to the uppermost liquid outlet channel 103a is the fourth liquid storage pool 201e-4.

The detection methods of CK-MB, Myo and cTnI are the same. You only need to replace the standard sample, the corresponding capture antibody, and the corresponding HRP-coupled detection antibody with those corresponding to the antigen.

The concentrations of capture antibodies for cTnI, CK-MB, and Myo myocardial markers during coating were 25 μg/mL, 20 μg/mL, and 40 μg/mL respectively. The concentrations of detection antibodies conjugated to HRP for cTnI, CK-MB, and Myo myocardial markers are 2.4 μg/mL, 2 μg/mL, and 3 μg/mL respectively.

5 pg/mL, 10 pg/mL, 20 pg/mL, 80 pg/mL, 320 pg/mL, 640 pg/mL, 1.28 ng/mL, 2.56 ng/mL, 5.12 ng/mL, 10.24 ng/ cTnI in the range of mL, 20.48 ng/mL, and 40.96 ng/mL was detected, and the results are shown in Figure 9. Fitting each point, as shown in Figure 10, shows that cTnI is within the range of 20 pg/mL, 80 pg/mL, 320 pg/mL, 640 pg/mL, 1.28 ng/mL, and 2.56 ng/mL. There is a good linear relationship.

5 pg/mL, 10 pg/mL, 20 pg/mL, 40 pg/mL, 80 pg/mL, 320 pg/mL, 640 pg/mL, 2.56 ng/mL, 5.12 ng/mL, 10.24 ng/ CK-MB was detected in the range of mL, 20.48 ng/mL, and 40.96 ng/mL, and the results are shown in Figure 9; by fitting each point, as shown in Figure 10, it can be obtained that CK-MB is at 80 pg /mL, 320 pg/mL, 640 pg/mL, 2.56 ng/mL, 5.12 ng/mL, and 10.24 ng/mL, showing a good linear relationship.

50 pg/mL, 100 pg/mL, 200 pg/mL, 400 pg/mL, 800 pg/mL, 6.4 ng/mL, 12.8ng/mL, 51.2 ng/mL, 102.4 ng/mL, 204.8 ng/ mL, 409.6 ng/mL, 819.2 ng/mL, Myo was detected in this range, and the results are shown in Figure 9; fitting each point, as shown in Figure 10, it can be obtained that Myo is in the range of 800 pg/mL, There is a good linear relationship in the range of 6.4ng/mL, 12.8 ng/mL, 51.2 ng/mL, 102.4 ng/mL, and 204.8 ng/mL.

The luminescence lattice for the separate detection of three myocardial markers, cTnI, CK-MB and Myo, is shown in Figure 11.

Example

This is the fifth embodiment of the present invention. The difference from Embodiments 1 to 4 is that this embodiment is a method for joint detection of three myocardial markers, cTnI, CK-MB, and Myo. The details are as follows:

Coat the reaction layer 203 with one capture antibody each of cTnI, CK-MB, and Myo. After assembling and preprocessing the chip, add 30 μL of standard sample to the first reservoir 201e-1, including cTnI and CK-MB. , 10 μL each of Myo; add 30 μL of detection antibodies coupled to HRP into the second reservoir 201e-2, in which 10 μL of detection antibodies coupled to HRP corresponding to cTnI, CK-MB, and Myo were added, and the initial concentrations were respectively are 7.2 μg/mL, 6 μg/mL, and 9 μg/mL; add 50 μL PBST washing solution to the third reservoir 201e-3, and add 35 μL Luminol-H to the fourth reservoir 201e-4. ₂ O ₂ chemiluminescence substrate;

Connect the negative pressure pump with a hose at the first negative pressure interface 201c and the second negative pressure interface 201d for driving the fluid in the chip;

The mechanical valve 103 is pressed down so that the lowermost liquid outlet channel 103a corresponds to the height of the liquid reservoir 201e. The first negative pressure pump operates to move the liquid in the first liquid reservoir 201e-1 and the second liquid reservoir 201e-2. The reagent is pumped into the mixing channel 201f. After the set time is reached, the first negative pressure pump stops and the second negative pressure pump operates to pump the mixed reagent from the other end of the mixing channel 201f to the direction of one end of the mixing channel 201f. The set time is reached. After a certain period of time, the second negative pressure pump stops operating, and the above operation is repeatedly cycled until the number of cycles reaches the set cycle number threshold, and the second negative pressure pump completely stops operating to obtain a uniformly mixed sample;

The first negative pressure pump operates to pump the evenly mixed sample into the reaction channel 201g. The first negative pressure pump stops operating and incubates statically for 15 minutes, allowing the mixture to specifically adsorb in the reaction channel 201g to form a double-antibody sandwich structure. ;

Afterwards, the first negative pressure pump continues to operate, sucking all the liquid in the reaction channel 201g into the waste liquid pool 201h;

Press down the mechanical valve 103 to make the middle liquid outlet channel 103a correspond to the position of the liquid reservoir 201e. The first negative pressure pump operates to allow the PBST washing liquid in the third liquid reservoir 201e-3 to enter the channel. This takes about 15 seconds. Continuous washing until all the PBST washing buffer enters the waste liquid pool for 201h, impurities not participating in the reaction and unbound HRP-coupled detection antibodies can be flushed into the waste liquid pool for 201h;

Continue to press the mechanical valve 103 to make the top liquid outlet channel 103a correspond to the position of the liquid reservoir 201e. The first negative pressure pump operates to cause the Luminol-H ₂ O ₂ chemiluminescent substrate to flow into the reaction channel 201g and connect with the detection antibody. HRP reaction, and then flowed into the waste liquid pool for 201h;

After the reaction process is completed, put the chip into the chemiluminescence detector and expose it for 20 s;

The three horizontal linear microchannels 201g-2 of the reaction channel 201g intersect with the three vertical capture antibody bands on the reaction layer 203. Each marker of cTnI, CK-MB, and Myo has three detection points, generating a total of nine detection points. The image is processed to obtain the chemiluminescence value of each point, and their average gray value minus the background value can be used to calculate the sample concentration.

The luminescent lattice for joint detection of three myocardial markers, cTnI, CK-MB and Myo, is shown in Figure 12.

It is not difficult to find that, without departing from the spirit and scope of the present invention, simple changes to the present invention, such as changing the chip structure, changing the reaction layer material, changing the detection markers, changing the type and dosage of the reagents, can achieve the expected purpose. Easy modifications and changes to the present invention are within the protection scope of the present invention.

It is important to note that the construction and arrangements of the present application shown in various exemplary embodiments are illustrative only. Although only a few embodiments are described in detail in this disclosure, those reviewing this disclosure will readily appreciate that many modifications are possible without materially departing from the novel teachings and advantages of the subject matter described in this application. is possible (e.g. changes in size, scale, structure, shape and proportion of various elements, as well as parameter values (e.g. temperature, pressure, etc.), mounting arrangements, use of materials, colors, orientations, etc.). For example, an element shown as integrally formed may be constructed from multiple parts or elements, the position of elements may be inverted or otherwise altered, and the nature or number or position of discrete elements may be altered or varied. Accordingly, all such modifications are intended to be included within the scope of this invention. The order or sequence of any process or method steps may be changed or reordered according to alternative embodiments. In the claims, any "means-plus-function" clause is intended to cover the structures described herein as performing the recited function and not only structural equivalents but also equivalent structures. Other substitutions, modifications, changes and omissions may be made in the design, operation and arrangement of the exemplary embodiments without departing from the scope of the invention. Therefore, the invention is not limited to particular embodiments, but extends to various modifications which still fall within the scope of the appended claims.

Furthermore, in order to provide a concise description of the exemplary embodiments, not all features of an actual implementation may be described (i.e., those features that are not relevant to the best mode presently contemplated for carrying out the invention, or that are not relevant to carrying out the invention) feature).

It is understood that numerous implementation-specific decisions may be made during the development of any actual implementation, as in any engineering or design project. Such a development effort might be complex and time consuming, but would be a routine undertaking of design, manufacture and production without undue experimentation to those of ordinary skill having the benefit of this disclosure .

It should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solution of the present invention can be carried out. Modifications or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of the claims of the present invention.

Claims (6)

1. A multi-marker quantitative detection method, characterized in that: it includes the following steps, At the same time, the detection sample and detection antibody are extracted into the mixing channel (201f) of the detection chip. The detection sample and detection antibody are circulated and pumped in the mixing channel (201f) to mix thoroughly. After the mixing is completed, the mixed solution enters the reaction channel (201g); After static incubation, the capture antibody coated on the reaction layer (203) corresponding to the position of the reaction channel (201g) specifically adsorbs the antigen, forming a double-antibody sandwich structure of capture antibody-antigen-detection antibody; Pump the reacted solution into the waste liquid pool (201h); The washing liquid washes the impurities that did not participate in the reaction and the unbound detection antibodies from the mixing channel (201f) and the reaction channel (201g) into the waste liquid pool (201h); The chemiluminescent substrate enters the reaction channel (201g) through the mixing channel (201f), and reacts with the detection antibody to produce chemiluminescence; The detection chip includes a chip assembly (200) and a control assembly (100). The chip assembly (200) includes a first chip body (201) and a second chip body (202) on the lower side of the first chip body (201). ), the downward end of the first chip body (201) is provided with several liquid reservoirs (201e), a mixing channel (201f) and a reaction channel (201g), and the reaction channel (201g) is far away from the liquid reservoir (201e ) The first chip body (201) at one end is provided with a first negative pressure interface (201c), and the first chip body (201) is provided with a number of injection ports that correspond to and are connected to the liquid reservoir (201e). The vent (201a) and the liquid reservoir (201e) can be connected to one end of the mixing channel (201f), and the other end of the mixing channel (201f) is connected to one end of the reaction channel (201g). The other end is connected to the waste liquid pool (201h). The waste liquid pool (201h) is connected to the first negative pressure interface (201c). There is a reaction device between the first chip body (201) and the second chip body (202). The reaction layer (203) corresponding to the position of the channel (201g), the first chip body (201) is provided with an upper valve hole (201b) that enables the liquid reservoir (201e) to communicate with one end of the mixing channel (201f); The control component (100) includes a mechanical valve (103), a first support bracket (102) and a second support bracket (101) connected together. The chip component (200) is supported on the first support bracket (102) and the second support bracket (101). Between the two support brackets (101), the second chip body (202) has a lower valve hole (202a) coaxial with the upper valve hole (201b), the first support bracket (102) and the second support bracket (102). There are first guide holes (102b) and second guide holes (101b) respectively on the upper valve hole (101). After the mechanical valve (103) passes through the first guide hole (102b) and is pressed into the upper valve hole (201b), the mechanical valve (103) The lower end of the valve (103) can pass through the lower valve hole (202a) and the second guide hole (101b) in sequence. The mechanical valve (103) is provided with a number of liquid outlet channels (103a) spaced in the height direction. , the liquid storage tank (201e) can be connected to one end of the mixing channel (201f) through the corresponding liquid outlet channel (103a), when the liquid storage tank (201e) is just right When connected, the liquid reservoir (201e) covers one end of the liquid outlet channel (103a), and one end of the mixing channel (201f) covers the other end of the liquid outlet channel (103a). 2. The method for quantitative detection of multiple markers according to claim 1, characterized in that: the first chip body (201) is also provided with a second negative pressure interface (201d), and the second negative pressure interface (201d) is 201d) is connected to one end of the mixing channel (201f). 3. The method for quantitative detection of multiple markers according to claim 1 or 2, characterized in that: the mechanical valve (103) is provided with a guide protrusion (103b), and the guide protrusion (103b) is connected to the mixing channel. (201f) When one end is aligned, the liquid inlet end of the bottom liquid outlet channel (103a) can correspond to multiple liquid reservoirs (201e), and the bottom liquid outlet channel (103a) has at least two liquid inlet ends. When the lower end of the lowermost liquid outlet channel (103a) is flush with the lower side of the first chip body (201), there are at least two liquid reservoirs (201e) covering different liquid inlet ends. 4. The multi-marker quantitative detection method according to claim 3, characterized in that: before detection, align the position of the guide protrusion (103b) with one end of the mixing channel (201f), and press down the mechanical valve (103) to make it Press into the upper valve hole (201b), and when the lowermost liquid outlet channel (103a) is pressed to the lower side of the first chip body (201), both sides of the liquid reservoir (201e) and the liquid outlet channel (103a) Corresponding to each liquid inlet end, stop pressing the mechanical valve (103), and inject the detection sample, detection antibody, washing solution and chemiluminescent substrate into different liquid storage pools (201e) respectively. At the first negative pressure interface (201c) The first negative pressure pump and the second negative pressure pump are respectively connected to the second negative pressure interface (201d). 5. The multi-marker quantitative detection method according to claim 1 or 2, characterized in that: when mixing the detection sample and the detection antibody, the first negative pressure pump operates first to pump the detection sample and the detection antibody into the mixing channel (201f ), when the pumped reagent approaches the other end of the mixing channel (201f), the first negative pressure pump stops and the second negative pressure pump works to move the mixed solution flowing to the other end of the mixing channel (201f) toward the mixing channel ( 201f) Flow in the direction of one end to complete one cycle of mixing. Repeat the above steps. When the number of cycle mixing reaches the set threshold, the second negative pressure pump stops and the first negative pressure pump sucks the mixed solution into the reaction channel. (201g) internal reaction. 6. The multi-marker quantitative detection method according to claim 3, characterized in that: after the reaction is completed, press the mechanical valve (103) downward to make the middle outlet channel (103a) connect with the storage solution injected with the washing liquid. Corresponding to the pool (201e), the first negative pressure pump operates, and the washing liquid flows through the outlet channel (103a), the mixing channel (201f) and the reaction channel (201g) in sequence and then rushes into the waste liquid pool (201h) to wash away the reaction layer. (203) to remove impurities and unbound detection antibodies that have not participated in the reaction; continue to press the mechanical valve (103) downward to connect the uppermost liquid outlet channel (103a) with the liquid reservoir (201e) injected with the chemiluminescent substrate. Correspondingly, the first negative pressure pump operates to pump the chemiluminescent substrate into the reaction channel (201g), react with the enzyme labeled on the detection antibody, and produce chemiluminescence; the horizontal linear microchannel (201g-) on the reaction channel (201g) 2) Form a luminescent lattice with the vertically coated antibody strips on the reaction layer (203), and measure the chemiluminescence value of each point.