CN113567421B - Quantitative detection method for multiple markers - Google Patents
Quantitative detection method for multiple markers Download PDFInfo
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- 238000002156 mixing Methods 0.000 claims abstract description 106
- 238000006243 chemical reaction Methods 0.000 claims abstract description 101
- 239000002699 waste material Substances 0.000 claims abstract description 32
- 238000005406 washing Methods 0.000 claims abstract description 24
- 239000000758 substrate Substances 0.000 claims abstract description 20
- 239000000243 solution Substances 0.000 claims abstract description 18
- 239000011259 mixed solution Substances 0.000 claims abstract description 16
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- 101100537532 Rattus norvegicus Tnni3 gene Proteins 0.000 description 21
- 239000012530 fluid Substances 0.000 description 10
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 9
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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Abstract
The application discloses a quantitative detection method for multiple markers, which comprises the following steps that a detection sample and a detection antibody are extracted and enter a mixing channel of a detection chip, the detection sample and the detection antibody are fully mixed in the mixing channel by circulating suction, and a mixed solution enters a reaction channel after the mixing is finished; standing and incubating, wherein the capture antibody coated on the reaction layer corresponding to the position of the reaction channel specifically adsorbs antigen to form a double-antibody sandwich structure of capture antibody-antigen-detection antibody; pumping the reacted solution into a waste liquid pool; washing liquid washes impurities which do not participate in the reaction of the mixing channel and the reaction channel and unbound detection antibodies into a waste liquid pool; the chemiluminescent substrate enters a reaction channel through a mixing channel and reacts with enzyme marked on the detection antibody to generate chemiluminescence; the application can realize synchronous sample injection of a plurality of solutions and has high detection efficiency.
Description
Technical Field
The application relates to the technical field of marker detection, in particular to a quantitative detection method for multiple markers.
Background
The instant test is a test mode which is performed on a sampling site and rapidly obtains a test result by using a portable analytical instrument and a matched reagent. Compared with traditional laboratory equipment, POCT equipment has equipment portability, detects fast, low to personnel's operation technical requirement, many-sided advantages such as maintenance of being convenient for. Especially for acute diseases, POCT technology is used for deep ambulance, family and community to carry out self early screening, which is a powerful means for guaranteeing health.
Enzyme-linked immunosorbent assay (ELISA) is a method based on the specific binding of antigen-antibody to this immune response. Based on ELISA, a double antibody sandwich method suitable for macromolecular proteins, a competition method suitable for small molecular proteins and other methods are developed. In the prior art, during detection, sample needs to be added step by step and washed for multiple times, so that the problems of long detection time and large reagent consumption exist, and the method has high technical requirements on operators.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present application has been made in view of the above-mentioned and/or existing problems in the detection of markers.
Therefore, the application aims to provide a multi-marker quantitative detection method which realizes synchronous sample injection, has short detection time, less reagent consumption and high detection efficiency.
In order to solve the technical problems, the application provides the following technical scheme: a multi-marker quantitative detection method comprises the following steps,
simultaneously extracting a detection sample and a detection antibody to enter a mixing channel of a detection chip, circularly sucking the detection sample and the detection antibody in the mixing channel to be fully mixed, and after the mixing is finished, allowing a mixed solution to enter a reaction channel;
standing and incubating, wherein the capture antibody coated on the reaction layer corresponding to the position of the reaction channel specifically adsorbs antigen to form a double-antibody sandwich structure of capture antibody-antigen-detection antibody;
pumping the reacted solution into a waste liquid pool;
washing liquid washes impurities which do not participate in the reaction of the mixing channel and the reaction channel and unbound detection antibodies into a waste liquid pool;
the chemiluminescent substrate enters the reaction channel through the mixing channel and reacts with the detection antibody to generate chemiluminescence.
As a preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: the detection chip comprises a chip assembly, the chip assembly comprises a first chip body and a second chip body at the lower side of the first chip body, one downward end of the first chip body is provided with a plurality of liquid reservoirs, a mixing channel and a reaction channel, a first negative pressure interface is arranged on the first chip body, which is far away from one end of the liquid reservoirs, a plurality of sample injection vent holes which are in one-to-one correspondence and are communicated with the liquid reservoirs are formed in the first chip body, the liquid reservoirs can be communicated with one end of the mixing channel, the other end of the mixing channel is connected with one end of the reaction channel, the other end of the reaction channel is connected with a waste liquid tank, the waste liquid tank is communicated with the first negative pressure interface, and a reaction layer which corresponds to the position where the reaction channel is located is arranged between the first chip body and the second chip body.
As a preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: the first chip body is provided with an upper valve hole which enables the liquid storage tank to be communicated with one end of the mixing channel.
As a preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: the detection chip further comprises a control assembly, the control assembly comprises a mechanical valve, a first support bracket and a second support bracket which are connected together, the chip assembly is supported between the first support bracket and the second support bracket, a lower valve hole coaxial with the upper valve hole is formed in the second chip body, a first guide hole and a second guide hole are formed in the first support bracket and the second support bracket respectively, and 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 sequentially pass through the lower valve hole and the second guide hole.
As a preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: the first chip body is also provided with a second negative pressure interface, and the second negative pressure interface is connected with one end of the mixing channel.
As a preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: the mechanical valve is provided with a plurality of liquid outlet channels which are arranged at intervals in the height direction, the liquid storage tank can be communicated with one end of the mixing channel through the corresponding liquid outlet channel, and when the liquid storage tank is just communicated with the mixing channel through the liquid outlet channel, the liquid storage tank 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 preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: the mechanical valve is provided with a guide bulge, when the guide bulge is aligned with one end of the mixing channel, the liquid inlet end of the liquid outlet channel at the bottom can correspond to a plurality of liquid storage tanks, the liquid outlet channel at the bottom is provided with at least two liquid inlet ends, and when the lower end of the liquid outlet channel at the bottom is aligned with the lower side of the first chip body, at least two liquid storage tanks respectively cover different liquid inlet ends.
As a preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: before detection, the guide bulge is aligned with the position of one end of the mixing channel, the mechanical valve is pressed into the upper valve hole by pressing down, when the lowest liquid outlet channel is pressed to the position of the lower side of the first chip body, the liquid storage tank corresponds to two liquid inlet ends of the liquid outlet channel, the mechanical valve is stopped from being pressed down, a detection sample, a detection antibody, washing liquid and chemiluminescent substrates are respectively injected into different liquid storage tanks, and the first negative pressure pump and the second negative pressure pump are respectively connected at the first negative pressure interface and the second negative pressure interface.
As a preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: when the detection sample and the detection antibody are mixed, the first negative pressure pump firstly acts to pump the detection sample and the detection antibody into the mixing channel, when the pumped reagent is close to the other end of the mixing channel, the first negative pressure pump stops acting, the second negative pressure pump works to flow the mixed solution flowing to the other end of the mixing channel towards the direction of one end of the mixing channel, one-time circulation mixing is completed, the steps are repeated, when the circulation mixing frequency reaches a set frequency threshold value, the second negative pressure pump stops acting, and the first negative pressure pump sucks the mixed solution into the reaction channel for reaction.
As a preferable scheme of the multi-marker quantitative detection method, the method comprises the following steps: after the reaction is finished, the mechanical valve is pressed downwards, so that the liquid outlet channel in the middle corresponds to the liquid storage tank filled with the washing liquid, the first negative pressure pump acts, the washing liquid is flushed into the waste liquid tank after passing through the liquid outlet channel, the mixing channel and the reaction channel in sequence, and impurities which do not participate in the reaction and unbound detection antibodies on the reaction layer are washed away; continuously pressing down the mechanical valve to enable the uppermost liquid outlet channel to correspond to the liquid storage tank filled with the chemiluminescent substrate, and enabling the chemiluminescent substrate to be pumped into the reaction channel by the first negative pressure pump to react with enzyme marked on the detection antibody to generate chemiluminescence; the transverse straight micro-channel on the reaction channel and the antibody strip vertically coated on the reaction layer form a luminous lattice, and the chemiluminescence values of all points are measured.
The application has the beneficial effects that: the application can realize the joint synchronous detection of a plurality of markers, and realize synchronous sample injection, and when different reagents are mixed, the reagents are fully and uniformly mixed by repeated circulation, so that the blockage in a mixing channel is avoided, the detection precision is improved, repeated washing is not needed, the reagent loss is less, and the detection cost is low.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
fig. 1 is a perspective view of the present application.
Fig. 2 is an exploded construction view of the present application.
Fig. 3 is a perspective view of a first chip body according to the present application.
FIG. 4 shows the flow rates of the solutions in the mixing channel and the reaction channel after mixing in the present application.
FIG. 5 is a graph showing the concentration change in the mixing channel and the reaction channel during the mixing of the solutions according to the present application.
FIG. 6 is a graph showing the concentration change in the mixing channel and the reaction channel after the completion of the mixing of the solutions in the present application.
FIG. 7 is a schematic diagram illustrating the detection principle of the present application.
FIG. 8 is a schematic illustration of the capture antibody coating of the reaction layer of the present application.
FIG. 9 is a graph showing the detection ranges of three myocardial markers, namely cTnI, CK-MB and Myo, according to the present application.
FIG. 10 is a linear graph showing the detection of three cardiac markers, cTnI, CK-MB and Myo, in the present application.
FIG. 11 is a luminous array chart of the application for independently detecting three myocardial markers of cTnI, CK-MB and Myo.
FIG. 12 is a luminous array chart of the application for joint detection of three myocardial markers, namely cTnI, CK-MB and Myo.
In the figure, a control component 100, a second support bracket 101a, a second connecting hole 101b, a second guiding hole 101b, a first support bracket 102a, a first connecting hole 102b, a first guiding hole 103, a liquid outlet channel 103a, a guiding protrusion 103b, a connecting pin 104, a chip component 200, a first chip body 201, a sample introduction vent hole 201a, a valve hole on 201b, a first negative pressure interface 201c, a second negative pressure interface 201d, a liquid storage tank 201e, a first liquid storage tank 201e-1, a second liquid storage tank 201e-2, a third liquid storage tank 201e-3, a fourth liquid storage tank 201e-4, a mixing channel 201f, a 201g reaction channel, a connecting micro-channel 201g-2, a linear micro-channel 201h waste liquid tank, a second chip body 202a valve hole, and a 203 reaction layer.
Description of the embodiments
Before describing the technical scheme of the present application, the terms used herein are defined as follows:
the term "PC" refers to: a polycarbonate;
the term "PDMS" refers to: polydimethyl siloxane;
the term "AMI" refers to: acute myocardial infarction;
the term "cTnI" refers to: cardiac troponin I;
the term "Myo" refers to: myoglobin;
the term "CK-MB" means: creatine kinase isozymes;
the term "HRP" refers to: horseradish peroxidase;
the term "BSA" refers to: bovine serum albumin;
the term "PBST" refers to: phosphate buffer containing 0.05% tween 20.
In order that the above-recited objects, features and advantages of the present application will become more readily apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. 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.
Examples
Referring to fig. 8, a first embodiment of the present application is a method for explaining the coating of a capture antibody and the pretreatment of a detection chip, comprising the steps of,
a PDMS micro-fluidic chip with a plurality of straight channels is vertically placed on the reaction layer 203, the channels are 500 mu m wide and 300 mu m deep, the gaps are 3 mm in parallel, the capture antibody solution is respectively injected into the channels, different channels are selected for injection according to the different quantity of detection markers, the reaction layer 203 is washed three times by using PBST washing liquid after 1 hour, the capture antibody which is not successfully coated is removed, and the capture antibody is coated on the reaction layer 203.
After the detection chip is assembled, 50 μl of 5% BSA solution is added into the first liquid storage tank 201e, the mechanical valve 103 is pressed to enable the lowest liquid outlet channel 103a to correspond to the height of the first liquid storage tank 201e, the BSA solution is pumped into the liquid outlet channel 103a until the whole channel is full, the incubation is carried out for 15 minutes at rest to seal the liquid outlet channel 103a and unoccluded vacant sites on the reaction layer 203, non-specific adsorption is avoided, the BSA solution is pumped into the waste liquid tank 201h of the detection chip completely, and the mechanical valve 103 is restored to the original position, so that the pretreatment of the chip is completed.
Examples
For the second embodiment of the application, the embodiment provides a multi-marker quantitative detection method which has high detection efficiency, less material consumption and detection cost reduction.
A quantitative detection method for multiple markers comprises the following steps,
simultaneously extracting a detection sample and a detection antibody to enter a mixing channel 201f of a detection chip, circularly sucking and fully mixing the detection sample and the detection antibody in the mixing channel 201f, and after the mixing is finished, allowing a mixed solution to enter a reaction channel 201g;
the reaction layer 203 corresponding to the 201g position of the reaction channel is subjected to static incubation, the capture antibody coated on the reaction layer is specifically adsorbed with antigen, and the antigen is specifically combined with the detection antibody, so that a double-antibody sandwich structure of the capture antibody, the antigen and the detection antibody is formed;
pumping the reacted solution into a waste liquid pool 201h;
washing liquid washes the impurities which do not participate in the reaction of the mixing channel 201f and the reaction channel 201g and the unbound detection antibody 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 generate chemiluminescence;
the chip assembly 200 is put into a chemiluminescent detection instrument for exposure, a luminescent lattice is formed by a transverse linear microchannel 201g-2 of a reaction channel 201g and an antibody strip vertically coated on a reaction layer 203, chemiluminescent values of all points are measured, and the average gray value of the chemiluminescent values minus a background value can be used for calculating the concentration of a sample.
Wherein the detection sample is a diluted serum sample or an antigen standard solution, the detection antibody is a detection antibody coupled with HRP, the washing liquid is PBST washing liquid, and the chemiluminescent substrate is Luminol-H 2 O 2 A chemiluminescent substrate.
Examples
Referring to fig. 1 to 3, a third embodiment of the present application is different from the first embodiment in that a specific structure of a detection chip is described, and when the detection chip is used in embodiment 2, supplementary explanation of steps related to embodiment 2 is provided.
The detection chip comprises a chip assembly 200, the chip assembly 200 comprises a first chip body 201 and a second chip body 202 at the lower side of the first chip body 201, one end of the first chip body 201 facing downwards is provided with a plurality of liquid storage tanks 201e, a mixing channel 201f and a reaction channel 201g, the reaction channel 201g comprises a plurality of sections of mutually parallel straight micro channels 201g-2, two adjacent straight micro channels 201g-2 are connected together through an arc-shaped connecting micro channel 201g-1, the straight micro channel 201g-2 at the head end is connected with the other end of the mixing channel 201f through the connecting micro channel 201g-1, the first chip body 201 of the reaction channel 201g far away from one end of the liquid storage tanks 201e is provided with a first negative pressure interface 201c, a through hole with the diameter of 1 mm penetrates through the first chip body 201, a plurality of sample introduction vent holes 201a which are in one-to-one correspondence with the liquid storage tanks 201e are arranged on the first chip body 201, the liquid storage tank 201e is formed by a rectangle with the length of 4 mm and the width of 3 mm and two semicircles with the diameter of 3 mm along the width direction, the depth of the liquid storage tank 201e is 3 mm, the total volume is about 60 mu L, the liquid storage tank 201e can be communicated with one end of a mixing channel 201f, an upper valve hole 201b which enables the liquid storage tank 201e to be communicated with one end of the mixing channel 201f is formed on the first chip body 201, the other end of the mixing channel 201f is connected with one end of a reaction channel 201g, one end of the mixing channel 201f gradually surrounds from outside to inside and then reversely surrounds from inside to outside to the other end of the mixing channel 201f, the radius gradually decreases when surrounding from outside to inside, specifically, the front half part of the mixing channel 201f is formed by connecting semicircles with the center of the mixing channel 201f with the radius of 7 mm, 5 mm, 3 mm and 1 mm respectively from the innermost end to outside, the latter half of the mixing channel 201f is formed by connecting the semicircle with the radius of 1 mm, 3 mm, 5 mm and 7 mm with the center of the mixing channel 201f, the other end of the reaction channel 201g is connected with the waste liquid tank 201h, the width of the reaction channel 201g is 400 μm, the depth is 400 μm, the distance between two adjacent straight micro channels 201g-2 is 3 mm, the length of the straight micro channel 201g-2 is 10 mm, the connecting micro channel 201g-1 is semicircle with the diameter of 3 mm, the waste liquid tank 201h is communicated with the first negative pressure interface 201c, a reaction layer 203 corresponding to the position of the reaction channel 201g is arranged between the first chip body 201 and the second chip body 202, the reaction layer 203 is 10 mm long, 10 mm wide, 0.1 mm thick silica gel plate, the waste liquid tank 201h is formed by the semicircle with the length of 20 mm, the rectangle with the width of 5 mm and the diameter of 5 mm in the width direction, the depth of the waste liquid tank 201h is 4 mm, the total volume is 400 μm, the total volume of the waste liquid tank 201g is used for holding the waste liquid in the detection process and the waste liquid can be used for carrying the apparatus 201 e.
Further, the detection chip further comprises a control assembly 100, the control assembly 100 comprises a mechanical valve 103, a first support bracket 102 and a second support bracket 101 which are connected together, the chip assembly 200 is supported between the first support bracket 102 and the second support bracket 101, both ends of the first support bracket 102 are provided with first connecting holes 102a, both ends of the second support bracket are provided with second connecting holes 101a, the first support bracket 102 and the second support bracket 101 are connected together through the first connecting holes 102a and the second connecting holes 101a by using a connecting pin 104, a lower valve hole 202a coaxial with the upper valve hole 201b is formed in the second chip body 202, a first guide hole 102b and a second guide hole 101b are formed in the first support bracket 102 and the second support bracket 101 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 sequentially pass through the lower valve hole 202a and the second guide hole 101b.
Further, the first chip body 201 is further provided with a second negative pressure interface 201d, the second negative pressure interface 201d and the first negative pressure interface 201c have the same structure, the second negative pressure interface 201d is connected with one end of the mixing channel 201f, the mechanical valve 103 is provided with a plurality of liquid outlet channels 103a which are arranged at intervals in the height direction, the liquid storage tank 201e can be communicated with one end of the mixing channel 201f through the corresponding liquid outlet channel 103a, when the liquid storage tank 201e is just communicated with the mixing channel 201f through the liquid outlet channel 103a, the liquid storage tank 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 103 a.
Further, 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 lowest liquid outlet channel 103a can correspond to the plurality of liquid storage tanks 201e, the lowest liquid outlet channel 103a has at least two liquid inlet ends, and when the lower end of the lowest liquid outlet channel 103a is aligned with the lower side of the first chip body 201, at least two liquid storage tanks 201e respectively cover different liquid inlet ends; by the arrangement of the guide protrusion 103b and one end of the mixing channel 201f, the mechanical valve 103 and the chip assembly 200 are circumferentially positioned for installation, and the installation is convenient.
Before detection by using the detection chip, the guide protrusion 103b is aligned with the position of one end of the mixing channel 201f, the mechanical valve 103 is pressed into the upper valve hole 201b, when the lowest liquid outlet channel 103a is pressed to the position of the lower side of the first chip body 201, two liquid storage tanks 201e containing detection samples and detection antibodies are respectively corresponding to two liquid inlet ends of the liquid outlet channel 103a, the mechanical valve 103 is stopped, the detection samples, the detection antibodies, washing liquid and chemiluminescent substrates are respectively injected into different liquid storage tanks 201e, and a first negative pressure pump and a second negative pressure pump are respectively connected at the first negative pressure interface 201c and the second negative pressure interface 201 d.
When the detection chip is used for mixing the detection sample and the detection antibody, the first negative pressure pump firstly acts to pump the detection sample and the detection antibody into the mixing channel 201f, and when the pumped reagent is close to the other end of the mixing channel 201f, the first negative pressure pump stops acting, the second negative pressure pump works to flow the mixed solution flowing to the other end of the mixing channel 201f towards the direction of one end of the mixing channel 201f, so that primary circulation mixing is completed; when the number of times of circulation mixing reaches the set number of times threshold, the number of times threshold is preferably 2 in this embodiment, the second negative pressure pump stops acting, the first negative pressure pump sucks the mixed solution into the reaction channel 201g for reaction, and referring to fig. 5 and 6, it can be seen that after continuous circulation mixing, the solution concentration is consistent, and uniform mixing of different samples is achieved.
After the reaction is finished, the mechanical valve 103 is pressed downwards, the middle liquid outlet channel 103a corresponds to the liquid storage tank 201e filled with the washing liquid, the first negative pressure pump acts, the washing liquid is flushed into the waste liquid tank 201h after passing through the liquid outlet channel 103a, the mixing channel 201f and the reaction channel 201g in sequence, and impurities which do not participate in the reaction and unbound detection antibodies on the reaction layer 203 are washed off; continuing to press the mechanical valve 103 downwards to enable the uppermost liquid outlet channel 103a to correspond to the liquid storage tank 201e filled with the chemiluminescent substrate, and enabling the chemiluminescent substrate to be pumped into the reaction channel 201g by the first negative pressure pump to react with enzyme marked on the detection antibody to generate chemiluminescence; the linear micro-channel 201g-2 on the reaction channel 201g and the antibody strip vertically coated on the reaction layer 203 form a light-emitting lattice, and the chemiluminescence values of each point are measured.
Referring to fig. 7, the pretreatment of the (a) chip includes coating a capture antibody on the re-reaction layer 203, blocking the hollow spot in the channel using BSA, and reagent loading; (B) The antigen and the detection antibody are mixed and reacted in the mixing channel 201 f; (C) A specific immune reaction occurs in the reaction channel 201g, and the capture antibody is specifically combined with the antigen; (D) washing the unreacted reagent; (E) adding a chemiluminescent substrate to produce a chemiluminescent signal.
In the detection method, the detection chip is used for detecting the marker, so that synchronous sample adding is realized, and the mixed solution after synchronous sample adding is uniformly mixed by circularly sucking the mixed solution during synchronous sample adding, so that the detection precision is improved; referring to fig. 4, the flow rates of the fluid movements of the mixing channel 201f and the reaction channel 201g are simulated by using multi-physical field simulation software, and the result shows that the fluid moves uniformly in the channel at the speed of 10 mm/s, and the conditions of fluid blockage, abrupt flow rate change, fluid distribution imbalance and the like do not occur, thus indicating that the fluid movement in the chip is stable.
Referring to fig. 6, in the case of the continuous circulation pumping mode in the mixing channel 201f and the specific structure of the mixing channel 201f, the mixing effect of the fluids in the present application is simulated by using the multi-physical field simulation software, and the result shows that the two fluids with different concentrations are mixed into the fluid with uniform concentration after passing through the mixing channel 201f, which indicates that the double-spiral micro-channels 1-4 have good mixing effect.
The application saves one-time incubation and washing operation, can finish quantitative detection of multiple markers within 17 min, and improves detection efficiency; the synchronous sample injection mixing of a plurality of reagents is realized, the full and uniform mixing of the mixed solution is realized through the combination of the chip assembly 200 and the detection method, the flow rate of the mixed solution in each channel is uniform, the detection under a plurality of concentrations is realized, and the detection is more reliable; the method is suitable for detection of most markers and has wide application range.
Examples
For the fourth embodiment of the present application, this embodiment is different from embodiments 1 to 3 in that the method for separately detecting three cardiac markers, cTnI, CK-MB, myo, comprises the steps of,
three cTnI capture antibodies are coated on the reaction layer 203, after the chip is assembled and pretreated, 30 mu L of cTnI standard sample is added to the first liquid storage tank 201e-1, 30 mu L of cTnI and HRP coupled detection antibody is added to the second liquid storage tank 201e-2, 50 mu L of PBST washing liquid is added to the third liquid storage tank 201e-3, and 35 mu L of Luminol-H is added to the fourth liquid storage tank 201e-4 2 O 2 A chemiluminescent substrate, and loading each reagent;
a negative pressure pump is connected with hoses at the first negative pressure interface 201c and the second negative pressure interface 201d and is used for driving fluid in the chip;
the mechanical valve 103 is pressed down, the liquid outlet channel 103a at the bottom corresponds to the height of the liquid storage tank 201e, the first negative pressure pump acts, the reagent in the first liquid storage tank 201e-1 and the second liquid storage tank 201e-2 is pumped into the mixing channel 201f, after a set time is reached, the first negative pressure pump stops acting, the second negative pressure pump acts, the mixed reagent is pumped from the other end of the mixing channel 201f to the direction of one end of the mixing channel 201f, after the set time is reached, the second negative pressure pump stops acting, the operation is repeatedly circulated, and when the circulation times reach a set circulation times threshold value, the second negative pressure pump completely stops acting, and a uniformly mixed sample is obtained;
the first negative pressure pump acts to pump the uniformly mixed sample into the reaction channel 201g, the first negative pressure pump stops acting and incubates for 15 minutes at rest, so that the mixed solution is subjected to specific adsorption in the reaction channel 201g to form a double-antibody sandwich structure;
then the first negative pressure pump continues to act, and all the liquid in the reaction channel 201g is sucked into the waste liquid pool 201h;
the pressing mechanical valve 103 is pressed to enable the middle liquid outlet channel 103a to correspond to the position of the liquid storage tank 201e, the first negative pressure pump is operated to enable PBST washing liquid in the third liquid storage tank 201e-3 to enter the channel, continuous washing is completed for about 15 seconds until all PBST washing buffer liquid enters the waste liquid tank 201h, and impurities which do not participate in reaction and unbound detection antibodies coupled with HRP can be flushed into the waste liquid tank 201h;
continuing to press down the mechanical valve 103 to enable the uppermost liquid outlet channel 103a to correspond to the position of the liquid storage tank 201e, and enabling the first negative pressure pump to act to enable Luminol-H to be discharged 2 O 2 The chemiluminescent substrate flows into the reaction channel 201g, reacts with HRP on the detection antibody, and then flows into the waste liquid pool 201h;
after the reaction process is finished, the chip is put into a chemiluminescent detector for exposure 20 s;
wherein, two reservoirs 201e communicating with the lowermost liquid outlet channel 103a are named as a first reservoir 201e-1 and a second reservoir 201e-2, respectively, the reservoir 201e communicating with the middle liquid outlet channel 103a is a third reservoir 201e-3, and the reservoir 201e communicating with the uppermost liquid outlet channel 103a is a fourth reservoir 201e-4.
The detection methods of CK-MB, myo and cTnI are consistent, and only the standard sample, the corresponding capture antibody and the corresponding HRP-coupled detection antibody are required to be replaced by the corresponding antigen.
The concentration of the capture antibodies of the cTnI, the CK-MB and the Myo myocardial markers when coated is 25 mug/mL, 20 mug/mL and 40 mug/mL respectively. The concentration of the detection antibody coupled with HRP of the three cardiac markers cTnI, CK-MB and Myo is 2.4 mug/mL, 2 mug/mL and 3 mug/mL respectively.
The cTnI was measured in the range of 5. 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/mL, 20.48 ng/mL, and 40.96 ng/mL, respectively, and the results are shown in FIG. 9. Fitting the points, as shown in FIG. 10, yields cTnI with good linear relationship in the range of 20 pg/mL, 80 pg/mL, 320 pg/mL, 640 pg/mL, 1.28 ng/mL, 2.56 ng/mL.
CK-MB in the range of 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/mL, 20.48 ng/mL, 40.96 ng/mL, respectively, were examined, and the results are shown in FIG. 9; the CK-MB is well linear in the ranges of 80 pg/mL, 320 pg/mL, 640 pg/mL, 2.56 ng/mL, 5.12 ng/mL and 10.24 ng/mL by fitting the points, as shown in FIG. 10.
Myo in this range was measured at 50. 50 pg/mL, 100 pg/mL, 200 pg/mL, 400 pg/mL, 800 pg/mL, 6.4 ng/mL, 12.8 ng/mL, 51.2 ng/mL, 102.4 ng/mL, 204.8 ng/mL, 409.6 ng/mL, 819.2 ng/mL, and the results are shown in FIG. 9; fitting the points, as shown in FIG. 10, yields Myo with good linearity over the range of 800 pg/mL, 6.4 ng/mL, 12.8 ng/mL, 51.2 ng/mL, 102.4 ng/mL, 204.8 ng/mL.
The luminescent dot matrix of the independent detection of three myocardial markers cTnI, CK-MB and Myo is shown in FIG. 11.
Examples
In the fifth embodiment of the present application, the difference from examples 1 to 4 is that this embodiment is a method for joint detection of three cardiac markers, namely cTnI, CK-MB, myo,
coating a capture antibody of cTnI, CK-MB and Myo on the reaction layer 203, assembling and preprocessing the chip, and adding 30 mu L of standard sample into the first liquid storage tank 201e-1, wherein each of the cTnI, CK-MB and Myo is 10 mu L; 30 mu L of HRP-conjugated detection antibody is added into the second liquid storage tank 201e-2, wherein 10 mu L of HRP-conjugated detection antibody corresponding to cTnI, CK-MB and Myo is added, and the initial concentration is 7.2 mu g/mL, 6 mu g/mL and 9 mu g/mL respectively; 50. Mu.L of PBST wash solution was added to each of the third reservoirs 201e-3, and 35. Mu.L of Luminol-H was added to the fourth reservoir 201e-4 2 O 2 A chemiluminescent substrate;
a negative pressure pump is connected with hoses at the first negative pressure interface 201c and the second negative pressure interface 201d and is used for driving fluid in the chip;
the mechanical valve 103 is pressed down, the liquid outlet channel 103a at the bottom corresponds to the height of the liquid storage tank 201e, the first negative pressure pump acts, the reagent in the first liquid storage tank 201e-1 and the second liquid storage tank 201e-2 is pumped into the mixing channel 201f, after a set time is reached, the first negative pressure pump stops acting, the second negative pressure pump acts, the mixed reagent is pumped from the other end of the mixing channel 201f to the direction of one end of the mixing channel 201f, after the set time is reached, the second negative pressure pump stops acting, the operation is repeatedly circulated, and when the circulation times reach a set circulation times threshold value, the second negative pressure pump completely stops acting, and a uniformly mixed sample is obtained;
the first negative pressure pump acts to pump the uniformly mixed sample into the reaction channel 201g, the first negative pressure pump stops acting and incubates for 15 minutes at rest, so that the mixed solution is subjected to specific adsorption in the reaction channel 201g to form a double-antibody sandwich structure;
then the first negative pressure pump continues to act, and all the liquid in the reaction channel 201g is sucked into the waste liquid pool 201h;
the pressing mechanical valve 103 is pressed to enable the middle liquid outlet channel 103a to correspond to the position of the liquid storage tank 201e, the first negative pressure pump is operated to enable PBST washing liquid in the third liquid storage tank 201e-3 to enter the channel, continuous washing is completed for about 15 seconds until all PBST washing buffer liquid enters the waste liquid tank 201h, and impurities which do not participate in reaction and unbound detection antibodies coupled with HRP can be flushed into the waste liquid tank 201h;
continuing to press down the mechanical valve 103 to enable the uppermost liquid outlet channel 103a to correspond to the position of the liquid storage tank 201e, and enabling the first negative pressure pump to act to enable Luminol-H to be discharged 2 O 2 The chemiluminescent substrate flows into the reaction channel 201g, reacts with HRP on the detection antibody, and then flows into the waste liquid pool 201h;
after the reaction process is finished, the chip is put into a chemiluminescent detector for exposure 20 s;
three horizontal straight micro-channels 201g-2 of the reaction channel 201g are intersected with three vertical capture antibody bands on the reaction layer 203, each marker of cTnI, CK-MB and Myo is provided with three detection points, nine detection points are formed by symbiotic, images are processed to obtain chemiluminescence values of all points, and the average gray value minus the background value can be used for calculating the concentration of a sample.
The luminescent lattice of the joint detection of three myocardial markers of cTnI, CK-MB and Myo is shown in figure 12.
It is readily apparent that the application can be simply modified, e.g., by changing the chip structure, changing the material of the reaction layer, changing the detected markers, changing the type and amount of the reagent to achieve the intended purpose without departing from the spirit and scope of the application, and that the application is easily modified and changed within the scope of the application.
It is important to note that the construction and arrangement of the application as shown in the various exemplary embodiments is illustrative only. Although only a few embodiments have been described in detail in this disclosure, those skilled in the art who review this disclosure will readily appreciate that many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters (e.g., temperature, pressure, etc.), mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter described in this application. For example, elements shown as integrally formed may be constructed of multiple parts or elements, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. Accordingly, all such modifications are intended to be included within the scope of present application. The order or sequence of any process or method steps may be varied or re-sequenced 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, operating conditions and arrangement of the exemplary embodiments without departing from the scope of the present applications. Therefore, the application is not limited to the specific embodiments, but extends to various modifications that nevertheless fall within the scope of the appended claims.
Furthermore, in order to provide a concise description of the exemplary embodiments, all features of an actual implementation may not be described (i.e., those not associated with the best mode presently contemplated for carrying out the application, or those not associated with practicing the application).
It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.
It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.
Claims (6)
1. A quantitative detection method for multiple markers is characterized in that: which comprises the steps of the following steps of,
simultaneously extracting a detection sample and a detection antibody to enter a mixing channel (201 f) of a detection chip, circularly sucking the detection sample and the detection antibody in the mixing channel (201 f), fully mixing, and after mixing, allowing a mixed solution to enter a reaction channel (201 g);
stationary incubation, wherein the capture antibody coated on the reaction layer (203) corresponding to the position of the reaction channel (201 g) specifically adsorbs antigen to form a double-antibody sandwich structure of capture antibody-antigen-detection antibody;
pumping the reacted solution into a waste liquid pool (201 h);
washing liquid washes impurities which do not participate in the reaction of the mixing channel (201 f) and the reaction channel (201 g) and unbound detection antibodies into a waste liquid pool (201 h);
the chemiluminescent substrate enters a reaction channel (201 g) through a mixing channel (201 f) and reacts with the detection antibody to generate chemiluminescence;
the detection chip comprises a chip assembly (200) and a control assembly (100), the chip assembly (200) comprises a first chip body (201) and a second chip body (202) which is arranged at the lower side of the first chip body (201), one downward end of the first chip body (201) is provided with a plurality of liquid storage tanks (201 e), a mixing channel (201 f) and a reaction channel (201 g), a first negative pressure interface (201 c) is arranged on the first chip body (201) which is far away from one end of the liquid storage tanks (201 e), a plurality of sample injection vent holes (201 a) which are in one-to-one correspondence with the liquid storage tanks (201 e) and are communicated with one another are formed in the first chip body (201), the liquid storage tanks (201 e) can be communicated with one end of the mixing channel (201 f), the other end of the mixing channel (201 f) is connected with one end of the reaction channel (201 g), the other end of the reaction channel (201 g) is connected with the waste liquid tank (201 h), the waste liquid tank (201 h) is communicated with the first negative pressure interface (201 c), a plurality of sample injection vent holes (201 a) which are in one-to-one end correspondence with the liquid storage tank (201 e), and the first chip body (201 f) is provided with one end of the first chip body (201 f), and the other end of the first chip body (201 f) is provided with one end of the liquid tank (201 h) and the liquid; the control assembly (100) comprises a mechanical valve (103), a first support bracket (102) and a second support bracket (101) which are connected together, the chip assembly (200) is supported between the first support bracket (102) and the second support bracket (101), a plurality of liquid outlet channels (103 a) which are arranged at intervals in the height direction are arranged on the second chip body (202), a first guide hole (102 b) and a second guide hole (101 b) are respectively arranged on the first support bracket (102) and the second support bracket (101), the mechanical valve (103) penetrates through the first guide hole (102 b) and is pressed into the upper valve hole (201 b), the lower end of the mechanical valve (103) can sequentially penetrate through the lower valve hole (202 a) and the second guide hole (101 b), the liquid storage tank (201 e) can be communicated with one end of the mixing channel (201 f) through the corresponding liquid outlet channels (103 a), and when the liquid storage tank (201 e) passes through the first guide hole (102 b) and is covered with one end of the mixing channel (201 e), and the other end of the liquid storage tank (201 e) is just communicated with one end of the mixing channel (201 e).
2. The method for quantitative detection of multiple markers according to claim 1, wherein: the first chip body (201) is also provided with a second negative pressure interface (201 d), and the second negative pressure interface (201 d) is connected with one end of the mixing channel (201 f).
3. The multi-marker quantitative detection method according to claim 1 or 2, characterized in that: the mechanical valve (103) is provided with a guide protrusion (103 b), when the guide protrusion (103 b) is aligned with one end of the mixing channel (201 f), the liquid inlet end of the lowest liquid outlet channel (103 a) can correspond to the liquid storage tanks (201 e), the lowest liquid outlet channel (103 a) is provided with at least two liquid inlet ends, and when the lower end of the lowest liquid outlet channel (103 a) is aligned with the lower side of the first chip body (201), at least two liquid storage tanks (201 e) respectively cover different liquid inlet ends.
4. The method for quantitative detection of multiple markers according to claim 3, wherein: before detection, the guide protrusion (103 b) is aligned with the position of one end of the mixing channel (201 f), the lower mechanical valve (103) is pressed into the upper valve hole (201 b), when the lowest liquid outlet channel (103 a) is pressed to the position of the lower side of the first chip body (201), the liquid storage tank (201 e) corresponds to the two liquid inlet ends of the liquid outlet channel (103 a), the lower mechanical valve (103) is stopped, a detection sample, a detection antibody, washing liquid and a chemiluminescent substrate are respectively injected into different liquid storage tanks (201 e), and the first negative pressure pump and the second negative pressure pump are respectively connected to the first negative pressure interface (201 c) and the second negative pressure interface (201 d).
5. The multi-marker quantitative detection method according to claim 1 or 2, characterized in that: when the detection sample and the detection antibody are mixed, the first negative pressure pump firstly acts to pump the detection sample and the detection antibody into the mixing channel (201 f), when the pumped reagent is close to the other end of the mixing channel (201 f), the first negative pressure pump stops acting, the second negative pressure pump works to flow the mixed solution flowing to the other end of the mixing channel (201 f) towards the direction of one end of the mixing channel (201 f), one-time circulation mixing is completed, the steps are repeated, when the circulation mixing frequency reaches a set frequency threshold value, the second negative pressure pump stops acting, and the first negative pressure pump sucks the mixed solution into the reaction channel (201 g) for reaction.
6. The method for quantitative detection of multiple markers according to claim 3, wherein: after the reaction is finished, the mechanical valve (103) is pressed downwards, so that the middle liquid outlet channel (103 a) corresponds to the liquid storage tank (201 e) filled with the washing liquid, the first negative pressure pump acts, the washing liquid is sequentially flushed into the waste liquid tank (201 h) through the liquid outlet channel (103 a), the mixing channel (201 f) and the reaction channel (201 g), and impurities which do not participate in the reaction and unbound detection antibodies on the reaction layer (203) are washed out; continuing to press the mechanical valve (103) downwards to enable the uppermost liquid outlet channel (103 a) to correspond to the liquid storage tank (201 e) filled with the chemiluminescent substrate, and enabling the chemiluminescent substrate to be pumped into the reaction channel (201 g) by the first negative pressure pump to react with enzyme marked on the detection antibody to generate chemiluminescence; a light-emitting lattice is formed by a transverse straight micro-channel (201 g-2) on the reaction channel (201 g) and an antibody strip vertically coated on the reaction layer (203), and the chemiluminescence values of all points are measured.
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