CN212068784U - Micro-fluidic paper chip for detecting human hypersensitive C reactive protein - Google Patents
Micro-fluidic paper chip for detecting human hypersensitive C reactive protein Download PDFInfo
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- CN212068784U CN212068784U CN202020025236.1U CN202020025236U CN212068784U CN 212068784 U CN212068784 U CN 212068784U CN 202020025236 U CN202020025236 U CN 202020025236U CN 212068784 U CN212068784 U CN 212068784U
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Abstract
The utility model discloses a micro-fluidic paper chip for detecting human hypersensitive C reactive protein, which comprises a substrate and a cover plate, wherein the substrate and the cover plate are formed by bonding, the cover plate is provided with a sample adding hole and a detection hole, the substrate is sequentially provided with a sample adding area, a reaction area, a detection area and a waste liquid area, the reaction area is coated with a hypersensitive C reactive protein antibody I marked by fluorescent microspheres, and the detection area is internally and fixedly coated with hypersensitive C reactive protein antibodies II with different epitopes; the utility model has high accuracy of the detection result, good linear relation, good stability, strong specificity and high sensitivity; the hypersensitive C reactive protein is the 'gold standard' for cardiovascular disease risk assessment, is a powerful predictive factor and risk factor for cardiovascular diseases, and can provide reliable clinical reference value for intervention and prognosis of cardiovascular diseases.
Description
Technical Field
The utility model relates to a micro-fluidic paper chip technical field specifically is a detect people's hypersensitivity C reaction protein and use micro-fluidic paper chip.
Background
C-reactive protein (CRP), which is found in serum of acute lobar pneumonia patients in 1930 by Tillet and Francis, is named as serum beta globulin with relative molecular mass of 115-140KD, which can perform precipitation reaction with C polysaccharide of pneumococcal cell wall in the presence of calcium ions. Research on CRP has been in history for over 70 years, and CRP is traditionally considered to be a non-specific marker of inflammation. CRP is mainly used for evaluating infection, tissue injury and inflammatory diseases clinically, provides data support for diagnosis of inflammatory diseases and provides diagnosis and treatment information for treatment and monitoring of diseases. With the advancement of detection technology, CRP detected using a hypersensitive method is called hypersensitive CRP (HS-CRP). In recent years, a large number of article researches show that HS-CRP plays an increasingly important role in diagnosis and prediction of diseases such as coronary heart disease, stroke, myocardial infarction, peripheral vascular embolism and the like. The role of CRP in the independent risk factors for cardiovascular disease caused by chronic inflammation has been demonstrated, and monitoring changes in CRP levels in time plays an important role in intervention and prognosis of cardiovascular disease, and the higher the CRP level, the greater the risk of cardiovascular and cerebrovascular events. Therefore, developed countries in Europe and America have used HS-CRP as a new relatively independent screening index for preventing cardiovascular diseases. The learners believe CRP is the "gold standard" for cardiovascular disease risk assessment, and is a powerful predictor and risk factor for cardiovascular disease.
At present, enzyme-linked immunoassay and colloidal gold colorimetric immune turbidimetry are adopted as detection methods of the hypersensitive C reactive protein: the colloidal gold colorimetric method has the characteristics of rapidness, simplicity, convenience and easy observation, but can only carry out qualitative or semi-quantitative detection, and has low sensitivity. The enzyme immunoassay method has simple operation and strong specificity, but has lower sensitivity and narrower linear range; the immunoturbidimetry is simple and convenient to operate, accurate and reliable in result, high in automation degree, rapid in detection and high in clinical usability. The micro-fluidic chip technology integrates basic operation units such as sample preparation, reaction, separation, detection and the like in the biological, chemical and medical analysis process on a micron-scale chip to automatically complete the whole analysis process; the operation process can be greatly simplified, the consumption of samples and reagents is reduced, expensive instruments are not required to be equipped, the on-site instant detection becomes possible, and the foreseeable huge economic value and social value are achieved; therefore, the establishment of a microfluidic chip technology platform for detecting the hypersensitive C reactive protein has very important significance.
SUMMERY OF THE UTILITY MODEL
Aiming at the defects of the prior art, the utility model provides a micro-fluidic paper chip for detecting human hypersensitive C reactive protein, which improves the accuracy and the sensitivity of detection.
In order to achieve the above object, the utility model provides a following technical scheme: a micro-fluidic paper chip for detecting human hypersensitive C reactive protein comprises a substrate and a cover plate, wherein the substrate and the cover plate are formed by bonding, the cross sections of the substrate and the cover plate are both rectangular structures, the cover plate is provided with sample adding holes and detection holes, the substrate is sequentially provided with a sample adding area, a reaction area, a detection area and a waste liquid area, the reaction area is coated with a hypersensitive C reactive protein antibody I marked by fluorescent microspheres, the detection area is internally and fixedly coated with a hypersensitive C reactive protein antibody II with different epitopes, the lower end of the sample adding area is provided with an opening communicated with the upper end of a filtering channel, the upper end of the reaction area is provided with an opening communicated with the lower end of the filtering channel, the filtering channel is internally provided with multilayer filter cotton, the lower end of the reaction area is provided with an opening communicated with the upper end of the reaction channel, the upper end of the detection area, the lower extreme of detection district is equipped with the opening and is linked together with the upper end of waste liquid passageway, and the upper end in waste liquid district is equipped with the opening and is linked together with the lower extreme of waste liquid passageway, be equipped with second current-limiting column in the waste liquid district, the cross-section of second current-limiting column is the arc structure, and second current-limiting column is located waste liquid district upper end opening part, the lower tip in waste liquid district is linked together with overflow channel's upper end.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the cross sections of the sample adding area, the reaction area, the detection area and the waste liquid area are all circular structures, and the diameters of the cross sections are the same.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the height of the waste liquid channel is in a descending trend from the detection area to the waste liquid area, and the highest point of the second flow limiting column is higher than the bottom of the detection area.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the filter cotton is a whole blood separation membrane.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the height of the overflow channel is lower than the highest point of the waste liquid channel.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the sample adding hole corresponds to the sample adding region, and the detection hole corresponds to the detection region.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the substrate and the cover plate are made of one of polydimethylsiloxane, polymethyl methacrylate and polytetrafluoroethylene.
The utility model discloses beneficial effect has: the utility model has the advantages of high sensitivity, good repeatability, extremely small sample amount, short detection time, simple operation, great simplification of operation process, reduction of sample and reagent consumption, no need of expensive instruments, possibility of on-site instant detection, and great economic value and social value; provides reliable clinical diagnosis value for intervention and prognosis of cardiovascular diseases.
Drawings
FIG. 1 is a schematic view of the bottom plate structure of the present invention;
FIG. 2 is a schematic view of the cover plate structure of the present invention;
FIG. 3 is a schematic sectional view taken along line A-A of FIG. 1;
FIG. 4 is a standard curve diagram of the detection of a hypersensitive C reactive protein standard;
in the figure: 1 base plate, 2 apron, 3 addition holes, 4 inspection holes, 5 addition district, 6 reaction zones, 7 detection areas, 8 waste liquid districts, 9 filtration passageways, 10 filter pulp, 11 reaction channel, 12 overflow passageways, 13 waste liquid passageways, 14 second current-limiting columns.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative work belong to the protection scope of the present invention.
Example 1
A micro-fluidic paper chip for detecting human hypersensitive C reactive protein comprises a substrate 1 and a cover plate 2, wherein the substrate 1 and the cover plate 2 are formed by bonding, the cross sections of the substrate 1 and the cover plate 2 are both rectangular structures, a sample adding hole 3 and a detection hole 4 are arranged on the cover plate 1, a sample adding area 5, a reaction area 6, a detection area 7 and a waste liquid area 8 are sequentially arranged on the substrate 1, the reaction area 6 is coated with a hypersensitive C reactive protein antibody I marked by fluorescent microspheres, a hypersensitive C reactive protein antibody II coated with different epitopes is fixedly arranged in the detection area 7, the lower end of the sample adding area 5 is provided with an opening communicated with the upper end of a filtering channel 9, the upper end of the reaction area 6 is provided with an opening communicated with the lower end of the filtering channel 9, a plurality of layers of filtering cotton 10 are arranged in the filtering channel 9, the lower end of the reaction area 6, the upper end of the detection area 7 is communicated with the lower end of the reaction channel 11, the reaction channel 11 is of an S-shaped structure, and the S-shaped structure of the reaction channel 11 can effectively control the time of the fluid in the reaction channel 11, so that the fluid to be detected and the antibody can fully and completely react; the lower extreme of detection zone 7 is equipped with the upper end that the opening is linked together with waste liquid channel 13, and the upper end of waste liquid district 8 is equipped with the lower extreme that the opening is linked together with waste liquid channel 13, be equipped with second current limiting column 14 in the waste liquid district 8, the cross-section of second current limiting column 14 is the arc structure, and second current limiting column 14 is located 8 upper end openings part in waste liquid district, and second current limiting column 14 can block the waste liquid in waste liquid district 8 and return detection zone 7, can play the effect of drainage simultaneously, and the fluid of detection zone 7 of being convenient for can flow into waste liquid district 8 smoothly, the lower tip of waste liquid district 8 is linked together with the upper end of overflow channel 12, and overflow channel is linked together with the micro-fluidic chip outside, effectively prevents the waste liquid adverse current.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the sections of the sample adding area 5, the reaction area 6, the detection area 7 and the waste liquid area 8 are all circular structures, and the diameters of the circular structures are the same.
Above-mentioned detect micro-fluidic paper chip for hypersensitivity C reaction protein of people, wherein, the height of waste liquid channel 13 is descending trend from the detection zone to the waste liquid district, the second current limiting post 14 highest point is higher than detection zone 7 bottom, and the fluid of the detection zone of being convenient for can flow into the waste liquid district smoothly, and can not flow back.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the filter cotton 10 is a whole blood separation membrane, the filter membrane is a whole blood separation membrane, a large pore in the membrane can intercept red blood cells and white blood cells in the whole blood in a physical mode, and plasma flows out from a small pore on the downstream surface of the membrane.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the height of the overflow channel is lower than the highest point of the waste liquid channel.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the sample adding hole 3 corresponds to the sample adding area 5, and the detection hole 4 corresponds to the detection area 7.
The microfluidic paper chip for detecting the human hypersensitive C reactive protein is characterized in that the substrate 1 and the cover plate 2 are made of one of polydimethylsiloxane, polymethyl methacrylate and polytetrafluoroethylene.
Example 2
The embodiment details the preparation method of the microfluidic chip for quantitatively detecting the hypersensitive C reactive protein, which specifically comprises the following steps:
(1) fixing the multilayer filter membrane in the reaction channel;
(2) washing fluorescent microspheres with 0.05mol/L MES buffer solution (pH 7.2), adding carbodiimide and N-hydroxysuccinimide after washing, reacting for 2h at room temperature to obtain a fluorescent microsphere solution, dissolving the hypersensitive C-reactive protein antibody I in 0.05mol/L PBS buffer solution (pH 7.2) to obtain a hypersensitive C-reactive protein antibody I solution, adding the fluorescent microsphere solution into the hypersensitive C-reactive protein antibody I solution, reacting for 2h at room temperature, centrifuging, filtering, washing for 3-5 times with 0.01mol/L PBS buffer solution (pH 7.2) to obtain a fluorescent microsphere labeled hypersensitive C-reactive protein antibody I, and dropwise coating the fluorescent microsphere labeled hypersensitive C-reactive protein antibody I on a reaction area;
(3) fixing and coating the hypersensitive C reactive protein antibody II in a detection area;
(4) the base plate is combined with the cover plate.
When in detection: adding the whole blood sample into the sample adding area from the sample adding hole, removing red blood cells and white blood cells of the whole blood sample through the filter cotton of the filter channel, and then entering the reaction area; reacting an antigen in a sample with a hypersensitivity C-reactive protein antibody marked by fluorescent microspheres to form an antigen-hypersensitivity C-reactive protein antibody-fluorescent microsphere complex, allowing the antigen-hypersensitivity C-reactive protein antibody I-fluorescent microsphere complex in a reaction region to enter a detection region from a reaction channel, and allowing the antigen-hypersensitivity C-reactive protein antibody I-fluorescent microsphere complex to perform specific immunoreaction with a hypersensitivity C-reactive protein antibody II fixed in the detection region to form a double-antibody sandwich complex of the fluorescent microsphere-hypersensitivity C-reactive protein antibody I-antigen-hypersensitivity C-reactive protein antibody II and fixing the double-antibody sandwich complex in the detection region; and (3) enabling an unfixed sample to enter a waste liquid area, adding a cleaning liquid through the sample adding hole, cleaning the microfluidic channel, and finally detecting the microfluidic chip through a fluorescence detector to detect the luminous intensity of the fluorescent microspheres, wherein the fluorescence intensity is related to the concentration of the object to be detected.
Example 3
The utility model discloses micro-fluidic chip performance analysis:
1. linearity: the utility model discloses use hypersensitivity C reactive protein standard concentration value (the standard is 0, 10, 25, 50, 100, 200ng/mL respectively) as the X axle to the standard signal value is the Y axle, establishes the calibration curve, calculates corresponding concentration value according to the signal value intensity of the sample that awaits measuring back, as shown in figure 4, linear equation is Y19.082X +151.34, R2 is 0.9955, and minimum detection limit is 0.8 ng/mL.
Taking a hypersensitive C reactive protein standard substance with the concentration of 25ng/ml to perform 10 times of tests, and calculating a detection concentration value by using a linear equation y which is 19.082x + 151.34; as shown in Table 1, the measured average values were 25.084ng/ml, the standard deviation was 314, and the intra-batch coefficient of variation was 1.3%.
TABLE 1
| Standard substance of hypersensitive C reactive protein (10pg/ml) | |
| For the first time | 25.14 |
| For the second time | 24.91 |
| The third time | 25.15 |
| Fourth time | 24.89 |
| Fifth time | 24.93 |
| The sixth time | 25.08 |
| The seventh time | 24.96 |
| The eighth time | 25.05 |
| The ninth time | 24.81 |
| The tenth time | 25.92 |
| Mean value of | 25.084 |
| Standard deviation of | 0.314 |
| Coefficient of variation within batch | 1.3％ |
2. Stability: taking the hypersensitive C reactive protein microfluidic chip to carry out a conventional storage stability test, and respectively detecting the hypersensitive C reactive protein microfluidic chip after being placed at the temperature of 2-8 ℃ for 1, 3, 5, 7, 9, 11, 12, 13, 14 and 15 months; the uncapping stability test is carried out according to the test of placing for 0 day, 7 days, 14 days, 16 days, 18 days, 24 days, 30 days, 35 days, 45 days and 60 days at the temperature of 2-8 ℃; the result shows that the hypersensitive C reactive protein micro-fluidic chip is stored in a dark environment at the temperature of 2-8 ℃ for 12 months, and is stored in a dark environment at the temperature of 2-8 ℃ for 45 days after being uncapped.
3. And (3) specific detection: the following endogenous substances were tested at the indicated concentrations, and the results are shown in table 2 below, without significant interference being observed.
TABLE 2
The utility model discloses the micro-fluidic chip testing result degree of accuracy is high, linear relation is good, stability is good, the specificity is strong, sensitivity is high, can provide reliable clinical reference value to intervention and prognosis of cardiovascular disease.
Various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims (3)
1. A micro-fluidic paper chip for detecting human hypersensitive C reactive protein is characterized by comprising a substrate and a cover plate, wherein the substrate and the cover plate are formed by bonding, the cross sections of the substrate and the cover plate are both rectangular structures, the cover plate is provided with a sample adding hole and a detection hole, the substrate is sequentially provided with a sample adding area, a reaction area, a detection area and a waste liquid area, the reaction area is coated with a hypersensitive C reactive protein antibody I marked by fluorescent microspheres, and the detection area is internally and fixedly coated with hypersensitive C reactive protein antibodies II with different epitopes; the lower end of the sample adding area is provided with an opening communicated with the upper end of the filtering channel, the upper end of the reaction area is provided with an opening communicated with the lower end of the filtering channel, the filtering channel is internally provided with a plurality of layers of filter cotton, the lower end of the reaction area is provided with an opening communicated with the upper end of the reaction channel, the upper end of the detection area is communicated with the lower end of the reaction channel, the reaction channel is of an S-shaped structure, the reaction channel is internally provided with a first flow limiting column, the lower end of the detection area is provided with an opening communicated with the upper end of the waste liquid channel, the upper end of the waste liquid area is provided with an opening communicated with the lower end of the waste liquid channel, the waste liquid area is internally provided with a second flow limiting column, the cross section; the sections of the sample adding area, the reaction area, the detection area and the waste liquid area are all circular structures, and the diameters of the circular structures are the same; the height of the waste liquid channel is in a descending trend from the detection area to the waste liquid area, and the highest point of the second flow limiting column is higher than the bottom of the detection area.
2. The microfluidic paper chip for detecting human hypersensitive C response protein according to claim 1, wherein said first flow restriction column has a cylindrical structure.
3. The microfluidic paper chip for detecting human hypersensitive C reactive protein according to claim 1, wherein said sample application well corresponds to a sample application region and said detection well corresponds to a detection region.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111111801A (en) * | 2020-01-07 | 2020-05-08 | 宁波奥丞生物科技有限公司 | Micro-fluidic paper chip for detecting human hypersensitive C reactive protein |
| CN113430106A (en) * | 2021-06-15 | 2021-09-24 | 皖南医学院 | Novel visual pathogen nucleic acid rapid detection chip |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111111801A (en) * | 2020-01-07 | 2020-05-08 | 宁波奥丞生物科技有限公司 | Micro-fluidic paper chip for detecting human hypersensitive C reactive protein |
| CN113430106A (en) * | 2021-06-15 | 2021-09-24 | 皖南医学院 | Novel visual pathogen nucleic acid rapid detection chip |
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