CN109001464B - Inflammation marker combined quantitative detection test paper and preparation method thereof - Google Patents
Inflammation marker combined quantitative detection test paper and preparation method thereof Download PDFInfo
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Abstract
The inflammation marker joint quantitative detection test paper comprises a sample pad, an inflammation marker antibody magnetic rare earth fluorescent microsphere label pad, a coating film and a water absorption pad, wherein three inflammation marker quantitative detection lines and a quality control region C line are arranged on the coating film, and after the detected substance in a sample is concentrated by a magnetic field, the detected substance is detected by using a low-background and high-sensitivity fluorescence immunochromatography method, so that the problem of low sensitivity of POCT products is solved. Can avoid the interference of other substances in the blood when the immune lateral chromatography reagent detects the blood sample.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to inflammation marker combined quantitative detection test paper based on using magnetic rare earth fluorescent microspheres as tracer markers and a preparation method thereof, in particular to a detection reagent card for determining the content of serum amyloid protein A/hypersensitive C reactive protein/procalcitonin in blood (serum, plasma and whole blood).
Background
Serum Amyloid A (SAA) is an acute phase-responsive Protein secreted mainly by hepatocytes, belonging to a heterogeneous class of proteins in the apolipoprotein family, with a relative molecular weight of about 12000. SAA is classified into acute-phase SAA (A-SAA) and constitutive SAA (C-SAA) according to the expression in vivo. SAA in normal human body is mainly derived from C-SAA expressed by liver cells in a constitutive mode, when the human body is stimulated by infection, inflammation, injury and the like, a series of cytokines are generated, and the level of the A-SAA is rapidly increased under the control of the cytokines to become the main SAA in the human body. The SAA level in the blood of the body can exceed 1000 times of the normal value in the reaction process of acute inflammation (such as trauma, infection and the like), and the characteristic makes the SAA one of the most sensitive inflammation markers at present.
Hypersensitive C-reactive protein (hs-CRP) is a C-reactive protein in blood plasma, and is also called hypersensitive C-reactive protein. The clinical guidance of the hypersensitive C reactive protein is mainly shown in the aspects of cardiovascular diseases, neonatal bacterial infection, kidney transplantation and the like. The hypersensitive C reactive protein is a nonspecific marker synthesized by the liver in the acute phase of systemic inflammatory response and is one of the most powerful predictors of the risk of cardiovascular events.
Procalcitonin (PCT) reflects the activity of systemic inflammatory responses, elevated plasma levels of PCT when severe bacterial, fungal, parasitic infections and sepsis and multi-organ failure, during which induction of bacterial endotoxin plays a crucial role. Whereas autoimmune, allergic and viral infections as well as locally limited bacterial infections, mild infections and chronic inflammation do not lead to elevated PCT levels in plasma. Various studies have shown that PCT is a good marker for bacterial infection and sepsis and an important tool in clinical diagnosis.
At present, the most widely used clinical diagnosis is to adopt imported reagents to detect serum amyloid A/hypersensitive C reactive protein/procalcitonin in serum and plasma. The existing kit on the market is complex in detection operation, time-consuming, not suitable for common screening and only can be used for a definite diagnosis test. Meanwhile, the sensitivity of the sample on the market is low because the sample is diluted and then tested.
Disclosure of Invention
In order to solve the problems and meet the requirement of conveniently testing the detection level of human serum amyloid A/hypersensitive C reactive protein/procalcitonin, the invention provides the inflammation marker combined quantitative detection test paper which is simple, reliable, low in cost and high in sensitivity; the detection method utilizes a magnetic field to concentrate the detected substance in the sample, and utilizes a low-background and high-sensitivity fluorescence immunochromatography to detect the detected substance, thereby solving the problem of low sensitivity of POCT products. Can avoid the interference of other substances in the blood when the immune lateral chromatography reagent detects the blood sample.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows: the inflammation marker combined quantitative detection test paper comprises a box body, wherein the box body comprises a bottom plate and a box cover, detection test paper is arranged in the box body, the bottom plate is arranged below the detection test paper, and the upper part of the detection test paper is connected with the box cover; the box cover is provided with a through hole area; the through hole area is provided with a sample adding hole and a result observation window; a detection area is arranged above the detection test paper and corresponds to the through hole area; the detection area comprises a sample pad, an inflammation marker antibody magnetic rare earth fluorescent microsphere labeling pad, a coating film and a water absorption pad, wherein three inflammation marker combined quantitative detection lines and a quality control area C line are arranged on the coating film, and the detection lines and the quality control area C line are arranged in parallel.
Further, the inflammation marker antibody magnetic rare earth fluorescent microsphere is formed by wrapping a magnetic rare earth fluorescent compound by a high polymer material, the sphere diameter of the magnetic rare earth fluorescent microsphere is 10-500nm, the fluorescence emission wavelength range is 400-800nm, and the surface modification active group of the nanoparticle is amino, carboxyl or sulfydryl.
Furthermore, two ends of the coating membrane are respectively connected with the water absorption pad and the inflammation marker antibody marking pad in an overlapping manner, and the coating membrane is a nitrocellulose membrane; a sample pad is pressed and stuck on the inflammation marker antibody marking pad, and the sample pad is a glass fiber sample pad.
Further, the inflammation marker antibody magnetic rare earth fluorescent microsphere label pad is glass fiber or polyester fiber.
A preparation method of inflammation marker combined quantitative detection test paper comprises the steps of inflammation marker antibody labeling and detection test paper preparation, wherein the inflammation marker antibody labeling step comprises the following steps:
firstly, coating a magnetic rare earth fluorescent microsphere prepared by Eu3+ -Fe3O4 compound by using a carboxylated polystyrene microsphere, wherein the particle size is 200nm, and the solid content is 1%;
adding 1mL of magnetic rare earth fluorescent microspheres into 5mL of pH 7.00.02M 3- (N-Malinio) propanesulfonic acid buffer solution (MOPS), uniformly mixing, centrifuging at 25000rpm for 10min, and discarding the supernatant; then adding 5mL of MOPS, performing 90W ultrasound, working for 2s, pausing for 5s, repeating for 2 times, centrifuging at 25000rpm for 10min, and removing the supernatant;
thirdly, adding 5mL of MOPS again, performing 90W ultrasonic treatment, working for 2s, performing intermittent operation for 5s, and repeating for 2 times to complete the cleaning of the microspheres;
adding 15mg of EDC and 50mg of sulfo-NHS into the cleaned microspheres, uniformly mixing by vortex, and reacting for 15min at 37 ℃;
fifthly, after the reaction is finished, adding 8.6 mu l of 2-mercaptoethanol to terminate the activation of carboxyl;
sixthly, respectively adding 1mg of serum amyloid protein A, hypersensitive C reactive protein and procalcitonin corresponding antibody, placing the mixture on a rotary incubator, and reacting for 90-120 min at 37 ℃;
seventhly, adding 100 mu L of 5% BSA and 100 mu L of 1% PEG20000, sealing unmarked sites, placing on a rotary incubator, and reacting at 37 ℃ for 60 min;
after reaction is finished, the liquid is centrifuged at 2000rpm for 15min, the supernatant is discarded, and a preservation solution (pH 8.00.2M boric acid borax buffer solution containing 1% BSA, 10% sucrose and 2% trehalose) is added into the supernatant for 5mL and is subjected to ultrasound for 2 s;
ninthly, diluting the marked microspheres by 50-100 times, and subpackaging each microsphere by 200 mu L to obtain a single-part semi-finished product.
The preparation method of the inflammation marker combined quantitative detection test paper comprises the following steps:
coating three detection lines of serum amyloid protein A, hypersensitive C reactive protein and antibodies corresponding to procalcitonin and a quality control line of goat anti-mouse/goat anti-rabbit IgG on a nitrocellulose membrane by using a BIODOT three-dimensional gold dot membrane spraying instrument according to the parameter of 1 mul/cm, and drying for 16 hours at 37 ℃ to serve as a reaction membrane.
② soaking the glass fiber by 0.1M PBS solution containing 0.5 percent Tween-20, then drying for 16 hours at 37 ℃, and cutting 17mm multiplied by 300mm to be used as a sample pad.
Thirdly, the sample pad, the reaction film and the absorbent paper (22 mm multiplied by 300 mm) are adhered and assembled on the bottom plate in a mode of laminating layer by layer.
Compared with the prior art, the invention has the following beneficial effects: the test strip comprises a sample pad, an inflammation marker antibody magnetic rare earth fluorescent microsphere marking pad, a coating film and a water absorption pad, wherein three inflammation marker combined quantitative detection lines and a quality control region C line are arranged on the coating film, a magnetic field is used for concentrating a detected substance in a sample, and then a low-background and high-sensitivity fluorescence immunochromatography method is used for detecting the detected substance, so that the problem of low sensitivity of POCT products is solved. Can avoid the interference of other substances in the blood when the immune lateral chromatography reagent detects the blood sample.
Drawings
FIG. 1 is a SAA test curve of the present invention;
FIG. 2 is a PCT test curve for the present invention;
FIG. 3 is a hs-CRP test curve according to the present invention;
FIG. 4 shows the results of the precision test of the SAA assay of the present invention;
FIG. 5 shows the results of the precision test of the PCT assay of the present invention;
FIG. 6 shows the results of the precision test of the hs-CRP measurement method of the present invention;
FIG. 7 is a schematic structural view of the present invention;
in the figure: 1. a base plate; 2. a sample pad; 3. an inflammation marker antibody magnetic rare earth fluorescent microsphere label pad; 4. a coating film; 5. a water absorbent pad; 6. a box body; 61. a box cover; 71. a sample application hole; 72. a result observation window; 8. an inflammation marker combined quantitative detection line; 9. and (4) a quality control area C line.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Unless the context clearly indicates otherwise, the names and parameters of the detection substances in the present invention may be present in a single form or in a plurality of forms, and the present invention is not limited thereto. Although the steps in the present invention are arranged by using reference numbers, the order of the steps is not limited, and the relative order of the steps can be adjusted unless the order of the steps is explicitly stated or other steps are required for the execution of a certain step. It is to be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
As can be seen from fig. 1-6, the inflammation marker combined quantitative detection test paper of the present invention comprises a box body, wherein the box body comprises a bottom plate and a box cover, the detection test paper is arranged in the box body, the bottom plate is arranged below the detection test paper, and the upper part of the detection test paper is connected with the box cover; the box cover is provided with a through hole area; the through hole area is provided with a sample adding hole and a result observation window; a detection area is arranged above the detection test paper and corresponds to the through hole area; the detection area comprises a sample pad, an inflammation marker antibody magnetic rare earth fluorescent microsphere labeling pad, a coating film and a water absorption pad, wherein three inflammation marker combined quantitative detection lines and a quality control area C line are arranged on the coating film, and the detection lines and the quality control area C line are arranged in parallel. The inflammation marker antibody magnetic rare earth fluorescent microsphere is formed by wrapping a magnetic rare earth fluorescent compound by a high polymer material, the sphere diameter of the magnetic rare earth fluorescent microsphere is 10-500nm, the fluorescence emission wavelength range is 400-800nm, and the surface modification active group of the nano microsphere is amino, carboxyl or sulfydryl. The two ends of the coating film are respectively connected with the water absorption pad and the inflammation marker antibody marking pad in an overlapping way, and the coating film is a nitrocellulose film; a sample pad is pressed and stuck on the inflammation marker antibody marking pad, and the sample pad is a glass fiber sample pad. The inflammation marker antibody magnetic rare earth fluorescent microsphere label pad is glass fiber or polyester fiber.
In the embodiment, the detection method is established as follows:
1.1 preparation of polystyrene microsphere seeds
1.1.1 weighing 100ml of styrene and a separating funnel, repeatedly washing with 1M NaOH for 3 times to remove impurities in the styrene, then washing with purified water to be neutral, and then placing in a drying dish for drying for more than 24 hours;
1.1.2 filling argon into a reaction device, and removing oxygen in the device;
1.1.3 adding 100ml of ethanol into a reaction device, then adding 5g of PVP, and stirring for 30 min;
1.1.4 adding 40ml of washed styrene and 5ml of Azobisisobutyronitrile (AIBN), heating to 45 ℃ in a water bath, reacting for 30min, then heating to 75 ℃ and reacting for 12 h;
1.1.5 after the reaction, filtering the reaction solution by using a mesh screen, washing and centrifuging for 3-5 times by using absolute ethyl alcohol, then putting the polymer subjected to preliminary impurity removal into 40% ethyl alcohol to naturally precipitate, and weighing the precipitate weight.
1.2 carboxylation modification of polystyrene surface
1.2.1 dispersing the polystyrene microspheres obtained by the above dispersion as seeds 1g with 10ml of 0.25% SDS;
1.2.2 adding cyclohexane of which the weight is 2 times that of the seeds into the system, and swelling for 16-18 h at 35 ℃;
1.2.3 weighing 0.02g of benzoyl peroxide, 10 mu l of ethylene glycol monomethyl ether and 10 mu l of ethylene glycol dimethacrylate, respectively adding the weighed materials into the reaction solution, and continuing swelling for 12 hours;
1.2.4, after swelling, adding 2.5mg of PVA, raising the reaction temperature to 75 ℃, and continuing to react for 10-12 hours;
1.2.5 repeated washing with ethanol 3 times to remove unreacted monomers and oligomers, followed by centrifugation at 20000rpm to settle the microspheres.
1.3 preparation of magnetic fluorescent nanospheres
1.3.1 FeCl2, FeCl3, EuCl3, NaOH solution, in molar proportions Fe2 +: fe3 +: eu3 +: and Na + =1:1:2:5, mixing the solution, stirring for reaction to obtain a precipitate, aging for 3-5 hours, filtering, washing with purified water for 2-3 times, drying, and grinding to obtain the Eu3+ -Fe3O4 compound.
1.3.2 adding 0.1g of carboxyl modified polystyrene microspheres into 20ml of isopropanol, and performing ultrasonic dispersion for 10 min;
1.3.3 weighing 0.5mg of Eu3+ -Fe3O4 compound, adding into 5ml of chloroform, and performing ultrasonic dispersion for 20 min;
1.3.4 mixing the two liquids of 1.3.2 and 1.3.3, placing the mixture in a vacuum environment for 5 to 6 hours, and then placing the mixture in normal pressure for 2 to 3 hours;
1.3.5 washing the swelled microsphere with ethanol for 3 times, and removing the microsphere which is not successfully wrapped with the magnetic particles by a magnetic field.
1.4 labeling of antibodies
1.4.1 taking 1ml of the prepared microspheres with the particle size of 200nm and the solid content of 1%, adding 5ml of 7.00.02M 3- (N-Malinio) propanesulfonic acid buffer solution (MOPS), uniformly mixing, centrifuging at 25000rpm for 10min, and discarding the supernatant;
1.4.2 adding 5ml MOPS, performing 90W ultrasound for 2s, intermittently repeating for 5s, repeating for 2 times, centrifuging at 25000rpm for 10min, and discarding the supernatant;
1.4.3, adding 5ml of MOPS again, performing 90W ultrasound, working for 2s, intermittently performing for 5s, repeating for 2 times, and finishing the cleaning of the microspheres;
1.4.4 adding 15mg of EDC and 50mg of sulfo-NHS into the cleaned microspheres, uniformly mixing by vortex, and reacting for 15min at 37 ℃;
1.4.5 after the reaction is finished, adding 8.6 mu l of 2-mercaptoethanol to terminate the activation of carboxyl;
1.4.6 adding 1mg of serum amyloid A, hypersensitive C reactive protein and antibodies corresponding to procalcitonin respectively, placing on a rotary incubator, and reacting for 90-120 min at 37 ℃;
1.4.7 then 100. mu.l of 5% BSA and 100. mu.l of 1% PEG20000 were added, the unlabelled sites were blocked, placed on a rotary incubator and reacted for 60min at 37 ℃;
1.4.8 after the reaction, centrifuging the above liquid at 2000rpm for 15min, discarding the supernatant, adding preserving solution (pH 8.00.2M borax borate buffer solution containing 1% BSA, 10% sucrose, 2% trehalose), 5ml, and performing ultrasound for 2 s;
1.4.9 diluting the marked microspheres by 50-100 times, and packaging each microsphere by 200 μ l to obtain semi-finished product, which is used in combination with test paper for determination of serum amyloid A/hypersensitive C reactive protein/procalcitonin.
1.5 preparation of test paper
1.5.1 coating three detection lines of serum amyloid protein A, hypersensitive C reactive protein and antibodies corresponding to procalcitonin and a quality control line of goat anti-mouse/goat anti-rabbit IgG on a nitrocellulose membrane by using a BIODOT three-dimensional gold-spraying membrane instrument according to the parameter of 1 mul/cm, and drying for 16 hours at 37 ℃ to be used as a reaction membrane.
1.5.2 soaking glass fiber with 0.1M PBS solution containing 0.5% Tween-20, then drying for 16 hours at 37 ℃, cutting 17mm × 300mm as sample pad.
1.5.3 the sample pad, the reaction membrane and the absorbent paper (22 mm x 300 mm) are adhered and assembled on the PVC base plate in a mode of laminating layer by layer, after cutting, the test paper for determining the serum amyloid A/the hypersensitive C reactive protein/the procalcitonin is obtained, and the test paper is put into a plastic card box, namely the reagent card for determining the serum amyloid A/the hypersensitive C reactive protein/the procalcitonin.
2. Evaluation of serum amyloid A/hypersensitive C reactive protein/Procalcitonin detection method
2.1 Linear Range
2.1.1, respectively diluting the high-value samples close to the upper limit of the linear range to at least 5 concentrations according to a certain proportion, wherein the low-value samples are required to be close to the lower limit of the linear range;
2.1.2 taking 100 mul of each concentration sample, respectively adding the samples into the packaged marked microspheres, and uniformly mixing by vortex;
2.1.3 placing the above reaction solution on a magnetic field, after 1min, discarding the supernatant, and then adding 200. mu.l PBS (0.02M, pH7.4) to each tube;
2.1.4 removing the magnetic field, uniformly mixing by vortexing again, respectively taking 100 mu l of each concentration sample, adding the samples into serum amyloid A/hypersensitive C reactive protein/procalcitonin test paper, and repeatedly testing for 3 times per concentration;
2.1.5 after 10min of reaction, placing the test paper in a dry immunofluorescence analyzer, reading the T/C value, calculating the T/C mean value of each concentration, fitting a concentration-reaction curve (the X axis is lg (concentration); the Y axis is lg (T/C)) by using a double-log straight line, wherein the SAA reaction curve is Y = 0.6839X-0.6532, and R =0.9928 as shown in the figure; PCT reaction curve y = 0.9431x-0.7463, R = 0.9988; the hs-CRP reaction curve is y = 1.07x-0.0506, R = 0.9954.
2.2 minimum detection Limit
Repeating the test for 20 times by operating the negative serum according to the ratio of 2.1.2-2.1.4, calculating the T/C mean value, and substituting the T/C mean value into a 2.1.5 test curve to obtain the lowest detection limit of the method, wherein the lowest detection limit of SAA is 5 mg/L; the PCT minimum detection limit is 0.1 ng/mL; the lowest detection limit of hs-CRP is 0.2 mg/L.
2.3 precision
The calibrator was diluted to three concentrations, high, medium and low, and the test was repeated 10 times per concentration, operating at 2.1.2-2.1.4. The results show that the coefficient of variation tested by the method is less than 10%, and therefore, the method has higher precision.
The method tests 87 parts of clinical serum, and the result of the serum amyloid protein A is as follows: y = 0.955x + 0.6172 with a correlation coefficient of 0.975, hypersensitive C response protein results: y = 0.9436x + 0.3112 with a correlation coefficient of 0.945, the procalcitonin result being: y = 0.9235x + 0.5312 with a correlation coefficient of 0.934.
Comprehensively, the method can detect serum amyloid A/hypersensitive C reactive protein/procalcitonin, has better precision and has the coefficient of variation less than 10 percent. Through clinical tests, the invention utilizes a magnetic field to concentrate the substance to be detected in a sample, and utilizes a low-background and high-sensitivity fluorescence immunochromatography to detect the substance to be detected, thereby solving the problem of low sensitivity of POCT products. The problem that the sensitivity is low due to the fact that the sample is diluted and then tested when the immune lateral chromatography reagent is used for detecting the blood sample can be solved, and interference of other substances in blood is reduced.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (1)
1. The preparation method of the inflammation marker combined quantitative detection test paper comprises the steps of inflammation marker antibody labeling and detection test paper preparation, wherein the inflammation marker antibody labeling step is as follows:
firstly, wrapping Eu by carboxylated polystyrene microspheres3+-Fe3O4The magnetic rare earth fluorescent microsphere prepared from the compound has the particle size of 200nm and the solid content of 1 percent;
adding 5mL of 0.02M 3- (N-Malineo) propanesulfonic acid buffer solution with pH of 7.0 into 1mL of magnetic rare earth fluorescent microspheres, uniformly mixing, centrifuging at 25000rpm for 10min, and removing supernatant; then adding 5mL of MOPS, performing 90W ultrasound, working for 2s, pausing for 5s, repeating for 2 times, centrifuging at 25000rpm for 10min, and removing the supernatant;
thirdly, adding 5mL of MOPS again, performing 90W ultrasonic treatment, working for 2s, performing intermittent operation for 5s, and repeating for 2 times to complete the cleaning of the microspheres;
adding 15mg of EDC and 50mg of sulfo-NHS into the cleaned microspheres, uniformly mixing by vortex, and reacting for 15min at 37 ℃;
fifthly, after the reaction is finished, adding 8.6 mu l of 2-mercaptoethanol to terminate the activation of carboxyl;
sixthly, respectively adding 1mg of serum amyloid protein A, hypersensitive C reactive protein and procalcitonin corresponding antibody, placing the mixture on a rotary incubator, and reacting for 90-120 min at 37 ℃;
seventhly, adding 100 mu L of 5% BSA and 100 mu L of 1% PEG20000, sealing unmarked sites, placing on a rotary incubator, and reacting at 37 ℃ for 60 min;
after the reaction is finished, centrifuging the liquid at 2000rpm for 15min, removing the supernatant, adding 5mL of preservation solution of borax borate buffer solution with the pH value of 8.00.2M and containing 1% BSA, 10% sucrose and 2% trehalose, and carrying out ultrasound for 2 s;
ninthly, diluting the marked microspheres by 50-100 times, and subpackaging 200 mu L of each microsphere to obtain a single-part semi-finished product;
the preparation method of the inflammation marker combined quantitative detection test paper comprises the following steps:
coating three detection lines of serum amyloid protein A, hypersensitive C reactive protein and antibodies corresponding to procalcitonin and a quality control line of goat anti-mouse/goat anti-rabbit IgG on a nitrocellulose membrane by using a BIODOT three-dimensional gold dot membrane spraying instrument according to the parameter of 1 mul/cm, and drying for 16 hours at 37 ℃ to serve as a reaction membrane;
soaking glass fiber in 0.1M PBS solution containing 0.5% Tween-20, drying at 37 deg.c for 16 hr, and cutting into 17mm × 300mm sample pad;
thirdly, sticking and assembling the sample pad, the reaction film and the absorbent paper with the thickness of 22mm multiplied by 300mm on a bottom plate in a mode of laminating layer by layer;
the preparation of the carboxylated polystyrene microspheres in the inflammation marker antibody marking step (I) is as follows:
1.1 preparation of polystyrene microsphere seeds
1.1.1 weighing 100ml of styrene, putting the styrene into a separating funnel, washing the styrene for 3 times by using 1M NaOH to remove impurities in the styrene, then washing the styrene to be neutral by using purified water, and then putting the styrene into a drying dish to dry for more than 24 hours;
1.1.2 filling argon into a reaction device, and removing oxygen in the device;
1.1.3 adding 100ml of ethanol into a reaction device, then adding 5g of PVP, and stirring for 30 min;
1.1.4 adding 40ml of washed styrene and 5ml of azobisisobutyronitrile, heating to 45 ℃ in a water bath, reacting for 30min, then heating to 75 ℃ and reacting for 12 h;
1.1.5 after the reaction is finished, filtering the reaction solution by using a mesh screen, washing and centrifuging for 3-5 times by using absolute ethyl alcohol, then putting the polymer subjected to preliminary impurity removal into 40% ethyl alcohol to naturally precipitate, and weighing the precipitate weight;
1.2 carboxylation modification of polystyrene surface
1.2.1 dispersing the polystyrene microspheres obtained by the above dispersion as seeds 1g with 10ml of 0.25% SDS;
1.2.2 adding cyclohexane of which the weight is 2 times that of the seeds into the system, and swelling for 16-18 h at 35 ℃;
1.2.3 weighing 0.02g of benzoyl peroxide, 10 mu l of ethylene glycol monomethyl ether and 10 mu l of ethylene glycol dimethacrylate, respectively adding the weighed materials into the reaction solution, and continuing swelling for 12 hours;
1.2.4, after swelling, adding 2.5mg of PVA, raising the reaction temperature to 75 ℃, and continuing to react for 10-12 hours;
1.2.5 repeatedly washing with ethanol for 3 times to remove unreacted monomers and oligomers, and then centrifugally settling microspheres at 20000 rpm;
in the step of marking the inflammation marker antibody, carboxylated polystyrene microspheres wrap Eu3+-Fe3O4The steps for preparing the magnetic rare earth fluorescent microsphere by the compound are as follows:
1.3.1 FeCl2、FeCl3、EuCl3NaOH solution, in molar proportion Fe2+：Fe3+：Eu3+：Na+Mixing the solution at a ratio of 1:1:2:5, stirring for reaction to obtain a precipitate, aging for 3-5 h, filtering, washing with purified water for 2-3 times, drying, and grinding to obtain Eu3+-Fe3O4And (c) a complex.
1.3.2 adding 0.1g of carboxyl modified polystyrene microspheres into 20ml of isopropanol, and performing ultrasonic dispersion for 10 min;
1.3.3 weighing Eu3+-Fe3O4Adding 0.5mg of the compound into 5ml of chloroform, and performing ultrasonic dispersion for 20 min;
1.3.4 mixing the two liquids of 1.3.2 and 1.3.3, placing the mixture in a vacuum environment for 5 to 6 hours, and then placing the mixture in normal pressure for 2 to 3 hours;
1.3.5 washing the swelled microsphere with ethanol for 3 times, and removing the microsphere which is not successfully wrapped with the magnetic particles by a magnetic field.
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| CN110609025A (en) * | 2019-10-09 | 2019-12-24 | 上海奥普生物医药有限公司 | Time-resolved fluorescence immunochromatographic kit for combined quantitative detection of CRP and SAA and preparation method thereof |
| CN111691220A (en) * | 2020-06-23 | 2020-09-22 | 杭州特种纸业有限公司 | In-vitro life detection material and preparation method thereof |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102302918A (en) * | 2011-06-13 | 2012-01-04 | 天津大学 | Magnetic fluorescent composite microsphere and method for preparing same |
| CN102565386A (en) * | 2011-12-29 | 2012-07-11 | 北京康美天鸿生物科技有限公司 | Magnetic fluorescent microsphere immunochromatography quantitative detection method |
| CN104949943A (en) * | 2014-03-25 | 2015-09-30 | 上海执诚生物科技有限公司 | Quick biological sample detection method |
| CN105548567A (en) * | 2016-01-18 | 2016-05-04 | 武汉菲恩生物科技有限公司 | Kit for time resolution fluorescent quantitative detection on PCT |
| CN106146642A (en) * | 2016-06-27 | 2016-11-23 | 浙江星博生物科技股份有限公司 | A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application |
| CN106771239A (en) * | 2016-12-16 | 2017-05-31 | 威海纽普生物技术有限公司 | Serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one to determine kit and preparation method |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2489105A1 (en) * | 2004-12-20 | 2006-06-20 | Yingliang Tian | Development of a method for employing polystyrene/dextran nabo-particles in immunoassay |
| CA2632261A1 (en) * | 2005-12-05 | 2007-06-14 | Guava Technologies | Particle-based analyte characterization |
| CN1975423A (en) * | 2006-09-06 | 2007-06-06 | 浙江清华长三角研究院 | Immuno magnetic bead and producing method, and method and test plate for detection |
| EP2212698B1 (en) * | 2007-11-14 | 2014-01-08 | Nigel Robert Caterer | Methods of quantification for lateral flow devices |
| CN104237522B (en) * | 2012-12-03 | 2016-12-21 | 武汉生之源生物科技股份有限公司 | Adiponectin reagent box for detecting content and preparation method thereof |
| CN104059204B (en) * | 2014-04-28 | 2017-04-05 | 苏州工业园区为真生物医药科技有限公司 | Surface cation magnetic polymer microsphere and its preparation method and application |
| CN105203768B (en) * | 2014-08-18 | 2017-01-18 | 董俊 | Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling |
| CN107238711B (en) * | 2017-05-18 | 2019-07-23 | 无锡市精神卫生中心 | A kind of diagnostic kit and its detection method detecting Alzheimer disease peripheral blood protein marker |
-
2018
- 2018-07-09 CN CN201810746660.2A patent/CN109001464B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102302918A (en) * | 2011-06-13 | 2012-01-04 | 天津大学 | Magnetic fluorescent composite microsphere and method for preparing same |
| CN102565386A (en) * | 2011-12-29 | 2012-07-11 | 北京康美天鸿生物科技有限公司 | Magnetic fluorescent microsphere immunochromatography quantitative detection method |
| CN104949943A (en) * | 2014-03-25 | 2015-09-30 | 上海执诚生物科技有限公司 | Quick biological sample detection method |
| CN105548567A (en) * | 2016-01-18 | 2016-05-04 | 武汉菲恩生物科技有限公司 | Kit for time resolution fluorescent quantitative detection on PCT |
| CN106146642A (en) * | 2016-06-27 | 2016-11-23 | 浙江星博生物科技股份有限公司 | A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application |
| CN106771239A (en) * | 2016-12-16 | 2017-05-31 | 威海纽普生物技术有限公司 | Serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one to determine kit and preparation method |
Non-Patent Citations (1)
| Title |
|---|
| Ultrasensitive protein detection: a case for microfluidic magnetic bead-based assays;Tekin H C , Gijs M A M;《Lab on a Chip》;20130903;第13卷(第24期);全文 * |
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