CN112858710A - NT-proBNP quantitative detection kit and preparation method thereof - Google Patents
NT-proBNP quantitative detection kit and preparation method thereof Download PDFInfo
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- CN112858710A CN112858710A CN202110202174.6A CN202110202174A CN112858710A CN 112858710 A CN112858710 A CN 112858710A CN 202110202174 A CN202110202174 A CN 202110202174A CN 112858710 A CN112858710 A CN 112858710A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N2035/00099—Characterised by type of test elements
- G01N2035/00158—Elements containing microarrays, i.e. "biochip"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
Abstract
A NT-proBNP quantitative detection kit and a preparation method thereof, comprising a chip and a reagent, wherein the chip comprises a chip substrate and a chip cover plate; a sample adding area, a reaction area, a detection area and a waste liquid pool are sequentially arranged on the chip substrate; the sample adding area is communicated with the reaction area through a filtering channel, and a blood filtering membrane is arranged in the filtering channel; the reaction zone is communicated with the detection zone through a reaction channel, a first flow limiting column is arranged in the reaction channel, the first flow limiting column is of a bent structure, and the bending direction extends along the width direction of the reaction channel; the detection area is communicated with the waste liquid pool through a waste liquid channel, a second flow limiting column is arranged at the communication position of the waste liquid channel and the waste liquid pool, the second flow limiting column is of a wave-shaped structure, and a bulge is arranged at the end, facing the waste liquid pool, of the second flow limiting column; the reaction zone is coated with NT-proBNP antibody I marked by fluorescent microspheres, and the detection zone is fixed with NT-proBNP antibody II; the chip cover plate is provided with a sample adding hole, a detection window and an air hole. The method has the advantages of quick and simple detection and capability of greatly improving the detection sensitivity and accuracy.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a quantitative detection kit for NT-proBNP (N-terminal brain natriuretic peptide precursor) and a preparation method thereof.
Background
Type B brain natriuretic peptide (B-type natural peptide) mainly comes from ventricle and is a good serum marker for judging the severity and prognosis of chronic heart failure. Normally only a small amount of BNP is stored in cardiomyocytes and its secretion depends on the activation of the BNP gene. When ventricular muscle is stretched or ischemic due to clinical relevant factors such as atrial fibrillation, heart valvular disease, ventricular volume load increase, pulmonary hypertension, size and function change of left and right ventricles, coronary heart disease and the like, BNP gene of cardiac muscle cells is activated to generate pre-BNP containing 134 amino acids, and signal peptide of the protein is cut into precursor peptide proBNP containing 108 amino acids; the precursor peptide is rapidly decomposed into BNP containing 32 amino acids and NT-proBNP containing 76 amino acids under the action of endonuclease. Left ventricular contraction is enhanced, ventricular volume load is increased, and cardiomyocyte expansion is a major factor in stimulating the production of BNP and NT-proBNP. The biological half-life of NT-BNP is 60-120 minutes, which is much longer than BNP with a biological half-life of only 20 minutes. In heart failure, the level of plasma BNP is obviously increased along with the severity of heart failure, the level of plasma NT-proBNP is correspondingly increased, and the increased proportion and absolute value of the plasma NT-proBNP exceed those of BNP, so that the NT-proBNP can be more sensitive as a marker of early-stage heart function damage.
NT-proBNP is an important cardiac function marker, is an important parameter for excluding diagnosis of acute and chronic heart failure clinically, and has wide application in the aspects of prevention screening, curative effect detection, prognosis evaluation, risk stratification and the like of heart failure patients. Especially under the background that the aging trend of China is more severe and the heart failure risk factor is increased to cause the heart failure morbidity to be continuously increased, the NT-proBNP is used for screening the high-risk population of the heart failure and has important significance for timely prevention, early discovery and effective management of the heart failure, thereby reducing the morbidity and mortality of the heart failure.
There are several methods for detecting NT-proBNP, and the current market for detecting NT-proBNP is immunological detection, which is more specific and sensitive. The common immunodetection methods mainly comprise: enzyme immunoassay, immunofluorescence, immunochemiluminescence, immuno-gold labeling, etc. The enzyme immunoassay method uses double monoclonal antibodies to detect the artificially synthesized NT-proBNP, is specific and has no cross reaction, but the method has complex operation and high cost and is not suitable for bedside rapid detection. The current enzyme-linked fluorescence method with a Meiriee VIDAS system in the market is commonly used, the linear range is 0-200ng/ml, and the analysis sensitivity is 0.05 ng/ml. The immunofluorescence method is a quantitative immunofluorescence detection method, two mouse monoclonal antibodies are combined with two different binding sites of NT-proBNP antigen, and one antibody is fluorescently labeled (tracer). The other is fixed on the wall of the test tube. The antibody reacts with NT-proBNP molecules in blood plasma or blood serum to form a sandwich compound, the fluorescence labeled antibody is bound on the wall of a test tube, the content of a fluorescence marker is counted by a luminescent reagent, the intensity of a fluorescence signal is in direct proportion to the concentration of the NT-proBNP of a sample, a standard sample is simultaneously measured, and the concentration of the NT-proBNP of the sample can be quantitatively obtained after a standard curve is prepared according to the NT-proBNP with known antigen concentration. The immunochemiluminescence method is a new method improved on the basis of two methods, namely a radioimmunoassay and an enzyme immunoassay, has no radioactivity and teratogen, has the advantages of the two methods, has long product validity period, no pollution to the environment and no toxicity or harm to human bodies, is an outstanding detection method of an in vitro diagnostic reagent at present, and has obvious advantages in technology, but has the defects of complex operation, long time consumption, high cost and unsuitability for rapid bedside detection.
The microfluidic chip technology integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes into a micron-scale chip, and automatically completes the whole analysis process. The microfluidic chip has the advantages of precise fluid control, small sample requirement, rapid reaction and large-scale integration, so that the microfluidic chip becomes a tool with great development potential for clinical diagnosis and disease screening. Therefore, establishing a microfluidic chip technology platform for detecting NT-proBNP has great significance.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the invention provides the NT-proBNP quantitative detection kit which is rapid, simple and convenient to detect and can greatly improve the detection sensitivity and accuracy.
In order to solve the technical problems, the invention adopts the technical scheme that: a quantitative detection kit for NT-proBNP comprises a chip and a reagent arranged on a chip functional area, wherein the chip comprises a chip substrate and a chip cover plate which are bonded; a sample adding area, a reaction area, a detection area and a waste liquid pool are sequentially arranged on the chip substrate; the sample adding area is communicated with the reaction area through a filtering channel, and a blood filtering membrane is arranged in the filtering channel; the reaction zone is communicated with the detection zone through a reaction channel, a first flow limiting column is arranged in the reaction channel, the first flow limiting column is of a bent structure, and the bending direction of the first flow limiting column extends along the width direction of the reaction channel; the detection area is communicated with the waste liquid pool through a waste liquid channel, a second flow limiting column is arranged at the communication position of the waste liquid channel and the waste liquid pool, the second flow limiting column is of a wave-shaped structure, and a bulge is arranged at the end, facing the waste liquid pool, of the second flow limiting column; the reaction zone is coated with NT-proBNP antibody I marked by fluorescent microspheres, and the detection zone is fixed with NT-proBNP antibody II; the chip cover plate is provided with a sample adding hole, a detection window and an air hole; the sample adding hole corresponds to the sample adding area, the detection window corresponds to the detection area, and the air hole is communicated with the outside.
By adopting the structure, the application designs a microfluidic chip structure suitable for quantitative detection of NT-proBNP, and combines reagents to form a complete NT-proBNP quantitative detection kit, the kit with the structure not only can accurately measure NT-proBNP, but also can adapt to microfluidic detection equipment, the detection is rapid and simple, and the detection sensitivity and accuracy can be greatly improved; the blood filtering membrane is arranged in the structure of the chip, so that blood plasma in whole blood can be rapidly separated without hemolysis, the influence of red blood cells on a detection result is avoided, and the interference on the detection result is reduced; the first flow limiting column is arranged in the structure of the chip, so that the flowing time of liquid can be controlled, and the liquid to be detected and the antibody can fully react; this application has set up second current-limiting post in the structure of chip, and the second current-limiting is the wave structure, has the arch towards waste liquid pond end, can play the drainage effect, makes liquid flow into the waste liquid pond smoothly to can prevent that the waste liquid refluence from returning the detection zone.
Preferably, the first flow limiting column is a strip, the length of the first flow limiting column extends along the direction of a reaction channel which is communicated with the reaction area and the detection area, the first flow limiting column is a bent shape formed by a plurality of cylindrical columns, gaps are arranged between adjacent cylindrical columns, and the medium flows through the gaps of the cylindrical column members; by adopting the structure, the medium can flow along the length direction of the first flow limiting column, and meanwhile, the gaps among the plurality of cylindrical columns can also flow the medium, so that the medium has the blocking force and the shearing force in a plurality of directions, the guarantee is provided for the better flow limiting of the medium, the flowing time of the medium is better controlled, and the liquid to be detected and the antibody can fully react.
Preferably, the first current-limiting column is provided with 5 bending parts, and the five bending parts are protruded in opposite directions in sequence; by adopting the structure, the time for the medium to flow along the first flow limiting column can be effectively controlled, and the guarantee is provided for the full reaction of the liquid to be detected and the antibody.
Preferably, the blood filtering membrane is filled in the filtering channel between the sample adding region and the reaction region and is mutually attached to the inner wall of the filtering channel, and the length of the blood filtering membrane is greater than half of the length of the filtering channel; by adopting the blood filtering membrane with the structure, the efficiency of separating the plasma in the whole blood is improved, and the plasma in the whole blood is fully, quickly and completely separated.
Preferably, the second flow limiting column is waved and extends along the width direction of the waste liquid channel, and a gap is arranged between the second flow limiting column and the chip cover plate and used for allowing a medium to flow out to the waste liquid pool; and the inside of the waste liquid channel is under a negative pressure condition so as to realize that the medium flows towards the waste liquid pool; adopt this structure, can effectual guarantee drainage effect, make liquid flow into the waste liquid pond smoothly to can prevent that the waste liquid refluence from returning the detection zone.
Preferably, the second flow-limiting column is located at the bottom end of the waste liquid channel, extends from the side of the chip substrate to the side of the chip cover plate in height, and is provided with a gap with the chip cover plate; the length of the waste liquid channel extends along the width direction of the bottom end of the waste liquid channel and is connected to two side walls in the width direction of the bottom end of the waste liquid channel; by adopting the structure, the second flow limiting column is a wavy platform structure, the medium is blocked in the waste liquid channel, and only the gap between the flow limiting column and the chip cover plate is reserved for medium flowing.
Preferably, the chip substrate and the chip cover plate are made of one of polymethyl methacrylate (PMMA), Polydimethylsiloxane (PDMS) and polytetrafluoroethylene.
Preferably, the axial direction of the cylindrical column is perpendicular to the flowing direction of the medium; i.e. the direction of axial extension or the direction of height extension of the cylindrical column is perpendicular to the axial direction of the reaction channel.
The above structure of the quantitative detection kit for NT-proBNP is a preferred embodiment of the present application, and reasonable variations within the structure range described above are considered to fall within the protection scope of the present application, and the quantitative detection kit for NT-proBNP is a microfluidic detection kit, and is suitable for microfluidic detection equipment.
The application also provides a preparation method of the NT-proBNP quantitative detection kit, which comprises the following specific steps:
(1) carrying out plasma treatment on the chip substrate to change the surface property of the chip substrate and increase the hydrophilicity;
(2) blocking redundant sites on the solid phase surface (referring to the whole chip surface) by using a blocking agent to prevent the antibody or the antigen from being non-specifically bound with the solid phase surface;
(3) marking the NT-proBNP antibody I by the fluorescent microsphere, and coating the NT-proBNP antibody I marked by the fluorescent microsphere in a reaction area;
(4) coating the NT-proBNP antibody II on a detection area;
(5) placing a blood filtration membrane in the filtration channel;
(6) bonding the chip substrate and the chip cover plate together;
(7) and (3) detection: adding a whole blood sample into the reaction area from the sample adding hole, filtering through a blood filtering membrane, and then enabling the sample to enter the reaction area; in the reaction zone, after the antigen in the sample reacts and is combined with the fluorescent microsphere labeled NT-proBNP antibody I, the antigen enters the detection zone; in the detection area, the compound is combined with NT-proBNP antibody II fixed in the detection area to form a double-antibody sandwich compound of NT-proBNP antibody I-NT-proBNP antigen-NT-proBNP antibody II, and is fixed in the detection area; and the sample which is not combined and fixed continues to flow into the waste liquid pool; and finally, detecting by a fluorescence detector, and reflecting the concentration of the sample to be detected by detecting the fluorescence intensity of the fluorescent microspheres connected with the NT-proBNP antibody I, wherein the fluorescence intensity is in positive correlation with the concentration of the sample to be detected.
Preferably, the method for labeling NT-proBNP antibody I by fluorescent microspheres in step (3) is as follows: diluting the fluorescent microsphere to 1mg/mL by using 0.05mol/L MES buffer solution (pH6.0), adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and reacting at room temperature for 30min to obtain an activated fluorescent microsphere solution; NT-proBNP antibody I was dissolved in 0.01mol/L PBS buffer (pH7.4) to obtain a solution of NT-proBNP antibody I with a concentration of 0.1mg/mL, and added to the above-mentioned fluorescent microsphere solution, reacted at room temperature for 2 hours, centrifuged and washed 3 times with 0.01mol/L PBS buffer (pH7.4) at which time NT-proBNP antibody I had bound to the fluorescent microspheres to form NT-proBNP antibody I-fluorescent microsphere complexes.
The application has the advantages and beneficial effects that:
1. the application is provided with the micro-fluidic chip aiming at the NT-proBNP for the first time, the chip can realize the rapid detection of the NT-proBNP, and meanwhile, the detection sensitivity and the accuracy are greatly improved.
2. The chip is applied to NT-proBNP detection, and has good repeatability and high stability.
3. In the chip structure, a first flow limiting column is arranged in a channel and is formed by combining a plurality of cylindrical columns, a gap is formed between every two adjacent columns for liquid (medium) to pass through, and the structure can control the flowing time of the liquid so as to ensure that the liquid to be detected and an antibody fully react; the detection zone rear end links to each other with waste liquid pond front end, be equipped with second current limiting column in the waste liquid pond, second current limiting column is the wave structure, this wave structure also presents wave curved form flat board or platform structure by monoblock cross-section, there is the arch towards waste liquid pond end, can play the drainage effect, this second current limiting column is close to and is provided with the clearance between the terminal surface of chip apron and the chip apron, make liquid (medium) flow into the waste liquid pond smoothly along this clearance, and can prevent that the waste liquid refluence from returning the detection zone.
4. In the chip structure of this application, be equipped with and strain the blood membrane, but plasma in the quickly separating whole blood and no hemolysis phenomenon avoid the influence of red blood cell to the testing result.
5. The application provides a micro-fluidic chip structure for NT-proBNP detection for the first time, and the structure realizes the purpose and effect of the micro-fluidic detection of NT-proBNP through a specific layout and distribution mode.
Drawings
Fig. 1 is a schematic structural diagram of a chip substrate according to the present invention.
Fig. 2 is a schematic structural diagram of the chip cover plate of the present invention.
Fig. 3 is a schematic view of the structure of the position of the first current-limiting column according to the present invention.
FIG. 4 is a schematic diagram of the structure of a cylindrical column constituting a first current-limiting column according to the present invention.
Fig. 5 is a schematic view of a second current-limiting post according to the present invention.
Fig. 6 is a schematic perspective view of a second current-limiting column according to the present invention.
FIG. 7 is a schematic diagram of a cross-sectional view of a second current-limiting column position of a chip according to the present invention.
Fig. 8 is a calibration curve of the present invention.
As shown in the figure: 1. the chip comprises a chip substrate, 2 parts of a chip cover plate, 3 parts of a sample adding area, 4 parts of a reaction area, 5 parts of a detection area, 6 parts of a waste liquid pool, 7 parts of a sample adding hole, 8 parts of a detection window, 9 parts of air holes, 10 parts of a filtering channel, 11 parts of a blood filtering membrane, 12 parts of a reaction channel, 13 parts of a first flow limiting column, 13.1 parts of a bending part, 14 parts of a waste liquid channel, 15 parts of a second flow limiting column, 15.1 parts of a bulge and 16 parts of a cylindrical column.
Detailed Description
The present invention will be described in detail below with reference to the following detailed description and accompanying drawings.
Examples
As shown in the attached drawings 1-2, the quantitative detection kit for NT-proBNP comprises a chip and a reagent arranged on a chip functional area, wherein the chip comprises a chip substrate 1 and a chip cover plate 2 which are bonded; a sample adding area 3, a reaction area 4, a detection area 5 and a waste liquid pool 6 are sequentially arranged on the chip substrate 1; the sample adding region 3 is communicated with the reaction region 4 through a filtering channel 10, and a blood filtering membrane 11 is arranged in the filtering channel 10; the reaction zone 4 is communicated with the detection zone 5 through a reaction channel 12, a first flow limiting column 13 is arranged in the reaction channel 12, the first flow limiting column 13 is of a bent structure, and the bending direction extends along the width direction of the reaction channel 12 (namely, the left side and the right side extend to form bent bulges); the detection area 5 is communicated with the waste liquid pool 6 through a waste liquid channel 14, a second flow limiting column 15 is arranged at the communication position of the waste liquid channel 14 and the waste liquid pool 6, the second flow limiting column is of a wave-shaped structure, and a bulge 15.1 is arranged at the end, facing the waste liquid pool, of the second flow limiting column; the reaction zone 4 is coated with NT-proBNP antibody I marked by fluorescent microspheres, and the detection zone 5 is fixed with NT-proBNP antibody II; the chip cover plate 2 is provided with a sample adding hole 7, a detection window 8 and an air hole 9; the sample adding hole 7 corresponds to the sample adding area 3, the detection window 8 corresponds to the detection area 5 in sequence, and the air hole 9 is communicated with the outside.
By adopting the structure, the application designs a microfluidic chip structure suitable for quantitative detection of NT-proBNP, and combines reagents to form a complete NT-proBNP quantitative detection kit, the kit with the structure not only can accurately measure NT-proBNP, but also can adapt to microfluidic detection equipment, the detection is rapid and simple, and the detection sensitivity and accuracy can be greatly improved; the blood filtering membrane is arranged in the structure of the chip, so that blood plasma in whole blood can be rapidly separated without hemolysis, the influence of red blood cells on a detection result is avoided, and the interference on the detection result is reduced; the first flow limiting column is arranged in the structure of the chip, so that the flowing time of liquid can be controlled, and the liquid to be detected and the antibody can fully react; this application has set up second current-limiting post in the structure of chip, the second current-limiting is the wave structure, there is the arch towards waste liquid pond end (wave structure is even arch from top to bottom, this embodiment is as shown in figure 4, 9 archs have been set up altogether, upwards have 5 towards waste liquid passageway direction promptly, there are 4 towards waste liquid pond direction promptly down, the structure of complete wave second current-limiting post is constituteed in the inter combination, can play the drainage effect, make liquid flow into the waste liquid pond smoothly, and can prevent that the waste liquid refluence from returning the detection zone.
As shown in FIGS. 1, 3-4, the first flow restriction column 13 is a column, the length of which extends along the direction of the reaction channel 12 communicating the reaction zone 4 and the detection zone 5 (specifically, as shown in FIGS. 1 and 3, the length of which can be as long as the reaction channel, so that the liquid flowing in the reaction channel can contact with the first flow restriction column and act on the medium), and the first flow restriction column is a curved shape composed of a plurality of cylindrical columns 13.1, and a gap is formed between adjacent cylindrical columns 13.1, and the medium flows between the gaps of the cylindrical columns; that is to say, the length extension direction of the first flow limiting column is arranged along the flowing direction of the medium in the reaction channel, so that the flowing path of the medium can be prolonged in the process of contacting with the first flow limiting column, the flow speed of the medium is effectively reduced, and the medium and the antibody in the reaction area are ensured to be more fully reacted; in addition, the application constitutes a complete first current-limiting column of a plurality of cylindrical columns, one end of which is connected with the bottom wall of the reaction channel, the other end of which is contacted with the chip cover plate, the medium only circulates in the gaps among the plurality of cylindrical columns; by adopting the structure, the medium can flow along the length direction of the first flow limiting column, so that the guarantee is provided for better flow limiting, and the flow time of the medium is better controlled, so that the liquid to be detected and the antibody can fully react; furthermore, more importantly, as shown in fig. 3, the first current-limiting column 13 of the present application is a curved shape composed of a plurality of cylindrical columns 16, gaps are formed between adjacent cylindrical columns, and the medium flows through the gaps of the cylindrical columns, so that the medium can be sheared to effectively reduce the flow speed, and meanwhile, the gaps in different directions generate multiple-angle shearing force to the medium, and the shearing force acts on the medium to ensure the sufficient mixing and contact of the medium.
As shown in fig. 1 and 3, the first current-limiting column 13 is provided with 5 bending parts 13.1, and the five bending parts are protruded in opposite directions in turn (i.e. as shown in fig. 1, the right-side bending protrusion of the first superterm is protruded, and the subsequent second is protruded toward the left side in turn according to the rule); namely, the first current-limiting column 13 in this embodiment is a curved S-shaped current-limiting column formed by sequentially connecting 5 bending parts, wherein the 5 bending parts form a wavy bending shape, and the bending directions are opposite and cross, and the bending or protruding directions are along the width or radial direction of the reaction channel; by adopting the structure, the time for the medium to flow along the first flow limiting column can be effectively controlled, and the guarantee is provided for the full reaction of the liquid to be detected and the antibody.
As shown in fig. 1, the blood filtering membrane 11 of the present application is filled in the filtering channel 10 between the sample adding region 3 and the reaction region 4, and is attached to the inner wall of the filtering channel 10, and the length of the blood filtering membrane 11 is greater than half of the length of the filtering channel; that is, the blood filtering membrane adopted in the embodiment occupies at least half of the length of the filtering channel, and the length of the contact path between the medium and the blood filtering membrane is increased; by adopting the blood filtering membrane with the structure, the efficiency of separating the plasma in the whole blood is improved, and the plasma in the whole blood is fully, quickly and completely separated.
As shown in fig. 1, 3-4, and 5-7 (which may be enlarged partial views), the second current-limiting column 15 is wavy and extends along the width direction of the waste liquid channel 14, and a gap is provided between the second current-limiting column 15 and the chip cover plate 2, and the gap is used for the medium to flow out to the waste liquid tank 6; and the inside of the waste liquid channel 14 is under negative pressure (that is, the inside of the chip is in a negative pressure state, and the medium flows towards the waste liquid pool through a siphon effect) so as to realize that the medium flows towards the waste liquid pool; as shown in fig. 6-7, or the second current-limiting column 15 of the present application is a waved platform structure, and the platform and the chip cover plate close to the end face are not closed (the chip cover plate is bonded with the chip substrate), and a gap is left, so that the medium (liquid) in the channel will flow out from the gap; adopt this structure, can effectual guarantee drainage effect, make liquid flow into the waste liquid pond smoothly to can prevent that the waste liquid refluence from returning the detection zone.
As shown in fig. 5-7, the second current-limiting column 15 is located at the bottom end of the waste liquid channel 14, extends from the chip substrate 1 side to the chip cover plate 2 side, and has a gap with the chip cover plate (i.e. a gap is provided between the end surface of the second current-limiting column facing the chip cover plate and the inner side wall of the chip cover plate, and the gap is used for medium to flow through); the length of the waste liquid channel extends along the width direction of the bottom end of the waste liquid channel and is connected to two side walls in the width direction of the bottom end of the waste liquid channel; by adopting the structure, the second flow limiting column is equivalent to a waved platform structure (as shown in figures 6 and 7), the medium is blocked in the waste liquid channel, and only the gap between the flow limiting column and the chip cover plate is reserved for medium flowing.
The size of the gaps among the plurality of cylindrical columns forming the specific shape of the flow-limiting column can ensure the size of flowing and circulating of the medium, and is suitable for the application, and the specific gaps can be adjusted according to the actual needs of the reaction; the size that medium flow through, circulation can be guaranteed to be provided with the clearance size between this application second current limiting column 15 and chip apron 2, all is adapted to this application.
The chip substrate and the chip cover plate are prepared from one of polymethyl methacrylate (PMMA), Polydimethylsiloxane (PDMS) and polytetrafluoroethylene. The chip prepared by adopting the materials has mature process, is convenient to process, and can effectively perform surface modification through a plasma treatment process to improve the hydrophilic property; the bonding of the chip substrate and the chip cover plate can be performed by adopting the most common bonding method for heat sealing, in addition, the methods such as anodic bonding and low-temperature bonding can also be selected, the conventional chip bonding method in the industry is adopted, the microfluidic channel can be performed by adopting the laser processing and other modes, and the details are not repeated.
The NT-proBNP quantitative detection kit is arranged on a micron-scale chip, and the specific flow channel structure of the chip is very critical, is suitable for the technical field of microfluidic chip detection, and can automatically complete the whole analysis process.
The NT-proBNP quantitative detection kit specifically comprises the following preparation steps:
(1) carrying out plasma treatment on the chip substrate to change the surface property of the chip substrate and increase the hydrophilicity;
(2) blocking redundant sites on the solid phase surface (referring to the whole chip surface) by using a blocking agent to prevent the antibody or the antigen from being non-specifically bound with the solid phase surface;
(3) marking the NT-proBNP antibody I by the fluorescent microsphere, and coating the NT-proBNP antibody I marked by the fluorescent microsphere in a reaction area;
the method for labeling the NT-proBNP antibody I by the fluorescent microspheres comprises the following steps: diluting the fluorescent microsphere to 1mg/mL by using 0.05mol/L MES buffer solution (pH6.0), adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and reacting at room temperature for 30min to obtain an activated fluorescent microsphere solution; NT-proBNP antibody I was dissolved in 0.01mol/L PBS buffer (pH7.4) to obtain a solution of NT-proBNP antibody I with a concentration of 0.1mg/mL, and added to the above-mentioned fluorescent microsphere solution, reacted at room temperature for 2 hours, centrifuged and washed 3 times with 0.01mol/L PBS buffer (pH7.4) at which time NT-proBNP antibody I had bound to the fluorescent microspheres to form NT-proBNP antibody I-fluorescent microsphere complexes.
(4) Coating the NT-proBNP antibody II on a detection area;
(5) placing the blood filtering membrane in a sample adding area filtering channel;
(6) bonding the chip substrate and the chip cover plate together;
(7) and (3) detection: adding a whole blood sample into the reaction area from the sample adding hole, filtering out red blood cells and the like through a blood filtering membrane, and then enabling the sample to enter the reaction area; in the reaction zone, after the antigen in the sample reacts and is combined with the fluorescent microsphere labeled NT-proBNP antibody I, the antigen enters the detection zone; in the detection area, the compound is combined with NT-proBNP antibody II fixed in the detection area to form a double-antibody sandwich compound of NT-proBNP antibody I-NT-proBNP antigen-NT-proBNP antibody II, and is fixed in the detection area; while the unbound sample continues to flow to the waste reservoir. And finally, detecting by a fluorescence detector, and reflecting the concentration of the sample to be detected by detecting the fluorescence intensity of the fluorescent microspheres connected with the NT-proBNP antibody I, wherein the fluorescence intensity is in positive correlation with the concentration of the sample to be detected.
And (3) analyzing the performance of the kit:
1. detection linearity: the kit takes concentration values of NT-proBNP standard substances (the standard substances are respectively 0pg/mL, 30pg/mL, 50pg/mL, 100pg/mL, 250pg/mL, 500pg/mL, 1000pg/mL, 10000pg/mL and 30000pg/mL) as an X axis, takes detection signal values of the standard substances as a Y axis (the detection signal values are shown in table 1), draws a scatter diagram, establishes a calibration curve and calculates corresponding concentration values according to the signal value intensity of a sample to be detected. As shown in fig. 8, the linear equation is y 1.3863x +700.72, R2The minimum detection limit was 0.9964 pg/mL.
2. Repeatability: as shown in table 2, the low value sample with a concentration value of 50pg/mL and the high value sample with a concentration value of 10000pg/mL of the NT-proBNP standard were tested 20 times, the detection concentration was calculated using the linear equation y of 1.3863x +700.72, and then the average value, standard deviation and CV value were calculated, and the obtained results show that the CV of the low value standard sample was 3.01% and the CV of the high value standard sample was 2.85%, indicating that the kit is good in performance.
3. Stability: taking the NT-proBNP quantitative detection kit for stability test, placing the NT-proBNP quantitative detection kit in a dark environment at 2-8 ℃, and respectively taking the NT-proBNP quantitative detection kit for testing after 0, 1, 3, 5, 7, 9, 10, 11, 12, 13, 14 and 15 months; the uncapping stability test is to put the NT-proBNP quantitative detection kit into a dark environment at 2-8 ℃ for detection with 0 day, 7 days, 14 days, 21 days, 28 days, 30 days, 32 days, 35 days, 40 days, 45 days, 55 days and 60 days. The result shows that the NT-proBNP quantitative detection kit is stored in a dark environment at the temperature of 2-8 ℃ and the effective period is 12 months; storing in a dark environment at 2-8 deg.C for 45 days.
TABLE 1 detection of concentration and luminescence values
TABLE 2 NT-proBNP standard sample with concentration value of 50pg/mL and high sample with concentration value of 10000pg/mL were tested repeatedly for 20 times
According to the detection data of the embodiment, the microfluidic NT-proBNP detection kit designed by the application has the advantages of good repeatability, high stability and accurate detection result.
Claims (10)
1. A NT-proBNP quantitative detection kit is characterized in that: the kit comprises a chip and a reagent arranged on a chip functional area, wherein the chip comprises a chip substrate and a chip cover plate which are bonded; a sample adding area, a reaction area, a detection area and a waste liquid pool are sequentially arranged on the chip substrate; the sample adding area is communicated with the reaction area through a filtering channel, and a blood filtering membrane is arranged in the filtering channel; the reaction zone is communicated with the detection zone through a reaction channel, a first flow limiting column is arranged in the reaction channel, the first flow limiting column is of a bent structure, and the bending direction of the first flow limiting column extends along the width direction of the reaction channel; the detection area is communicated with the waste liquid pool through a waste liquid channel, a second flow limiting column is arranged at the communication position of the waste liquid channel and the waste liquid pool, the second flow limiting column is of a wave-shaped structure, and a bulge is arranged at the end, facing the waste liquid pool, of the second flow limiting column; the reaction zone is coated with NT-proBNP antibody I marked by fluorescent microspheres, and the detection zone is fixed with NT-proBNP antibody II; the chip cover plate is provided with a sample adding hole, a detection window and an air hole; the sample adding hole corresponds to the sample adding area, the detection window corresponds to the detection area, and the air hole is communicated with the outside.
2. The quantitative detection kit for NT-proBNP according to claim 1, wherein: the first flow limiting column is one, the length of the first flow limiting column extends along the direction of a reaction channel which is communicated with the reaction area and the detection area, the first flow limiting column is in a bent shape and consists of a plurality of cylindrical columns, gaps are formed between every two adjacent cylindrical columns, and the medium flows through the gaps of the cylindrical columns.
3. The quantitative detection kit for NT-proBNP according to claim 1, wherein: first current-limiting post be provided with 5 kink, and five kink opposite direction projections in proper order.
4. The quantitative detection kit for NT-proBNP according to claim 1, wherein: the blood filtering membrane is filled in the filtering channel between the sample adding area and the reaction area and is mutually attached to the inner wall of the filtering channel, and the length of the blood filtering membrane is more than half of the length of the filtering channel.
5. The quantitative detection kit for NT-proBNP according to claim 1, wherein: the second flow limiting column is wavy and extends along the width direction of the waste liquid channel, and a gap is formed between the second flow limiting column and the chip cover plate and used for allowing a medium to flow out of the waste liquid tank; and the inside of the waste liquid channel is under a negative pressure condition so as to realize that the medium flows towards the waste liquid pool.
6. The quantitative detection kit for NT-proBNP according to claim 5, wherein: the second flow limiting column is positioned at the bottom end of the waste liquid channel, extends from the side of the chip substrate to the side of the chip cover plate in height, and is provided with a gap with the chip cover plate; the length of the waste liquid channel extends along the width direction of the bottom end of the waste liquid channel and is connected to two side walls of the width direction of the bottom end of the waste liquid channel.
7. The quantitative detection kit for NT-proBNP according to claim 1, wherein: the chip substrate and the chip cover plate are prepared from one of polymethyl methacrylate, polydimethylsiloxane and polytetrafluoroethylene.
8. The quantitative detection kit for NT-proBNP according to claim 2, wherein: the axial direction of the cylindrical column is perpendicular to the flowing direction of the medium.
9. A method for preparing the NT-proBNP kit according to any one of claims 1 to 8, wherein: the method comprises the following specific steps:
(1) carrying out plasma treatment on the chip substrate to change the surface property of the chip substrate and increase the hydrophilicity;
(2) blocking redundant sites on the solid phase surface by using a blocking agent to prevent the antibody or the antigen from being non-specifically combined with the solid phase surface;
(3) marking the NT-proBNP antibody I by the fluorescent microsphere, and coating the NT-proBNP antibody I marked by the fluorescent microsphere in a reaction area;
(4) coating the NT-proBNP antibody II on a detection area;
(5) placing a blood filtration membrane in the filtration channel;
(6) bonding the chip substrate and the chip cover plate together;
(7) and (3) detection: adding a whole blood sample into the reaction area from the sample adding hole, filtering through a blood filtering membrane, and then enabling the sample to enter the reaction area; in the reaction zone, after the antigen in the sample reacts and is combined with the fluorescent microsphere labeled NT-proBNP antibody I, the antigen enters the detection zone; in the detection area, the compound is combined with NT-proBNP antibody II fixed in the detection area to form a double-antibody sandwich compound of NT-proBNP antibody I-NT-proBNP antigen-NT-proBNP antibody II, and is fixed in the detection area; and the sample which is not combined and fixed continues to flow into the waste liquid pool; and finally, detecting by a fluorescence detector, and reflecting the concentration of the sample to be detected by detecting the fluorescence intensity of the fluorescent microspheres connected with the NT-proBNP antibody I, wherein the fluorescence intensity is in positive correlation with the concentration of the sample to be detected.
10. The method for preparing the NT-proBNP kit according to claim 9, wherein: the method for marking NT-proBNP antibody I by the fluorescent microspheres in step (3) is as follows: diluting the fluorescent microsphere to 1mg/mL by using 0.05mol/L MES buffer solution (pH6.0), adding carbodiimide and N-hydroxysuccinimide, and reacting at room temperature for 30min to obtain an activated fluorescent microsphere solution; NT-proBNP antibody I was dissolved in 0.01mol/L PBS buffer (pH7.4) to obtain a solution of NT-proBNP antibody I with a concentration of 0.1mg/mL, and added to the above-mentioned fluorescent microsphere solution, reacted at room temperature for 2 hours, centrifuged and washed 3 times with 0.01mol/L PBS buffer (pH7.4) at which time NT-proBNP antibody I had bound to the fluorescent microspheres to form NT-proBNP antibody I-fluorescent microsphere complexes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108686727A (en) * | 2018-06-26 | 2018-10-23 | 宁波奥丞生物科技有限公司 | The micro-fluidic chip of Quantitative detection PLGF and sFLT-1 |
| CN113430106A (en) * | 2021-06-15 | 2021-09-24 | 皖南医学院 | Novel visual pathogen nucleic acid rapid detection chip |
| CN116042382A (en) * | 2022-11-25 | 2023-05-02 | 山东第一医科大学(山东省医学科学院) | Multi-target gene joint detection sample boat |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108686727A (en) * | 2018-06-26 | 2018-10-23 | 宁波奥丞生物科技有限公司 | The micro-fluidic chip of Quantitative detection PLGF and sFLT-1 |
| CN113430106A (en) * | 2021-06-15 | 2021-09-24 | 皖南医学院 | Novel visual pathogen nucleic acid rapid detection chip |
| CN116042382A (en) * | 2022-11-25 | 2023-05-02 | 山东第一医科大学(山东省医学科学院) | Multi-target gene joint detection sample boat |
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