CN111504959B - C peptide rare earth fluorescent microsphere kit, detection card and preparation method thereof - Google Patents
C peptide rare earth fluorescent microsphere kit, detection card and preparation method thereof Download PDFInfo
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Abstract
The utility model discloses a C peptide rare earth fluorescent microsphere kit, a detection card and a preparation method thereof, wherein the detection card comprises a card shell and a detection test strip arranged in the card shell; the detection test strip comprises a bottom lining, a sample pad, a bonding pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the bonding pad, the nitrocellulose membrane and the absorbent paper are sequentially lapped and stuck on the bottom lining along the length direction; the binding pad is sprayed with a microsphere line formed by a C peptide monoclonal antibody marked by a C peptide rare earth fluorescent microsphere; the rare earth fluorescent microsphere is a rare earth ion complex doped fluorescent microsphere; the nitrocellulose membrane is provided with a detection line and a quality control line in parallel; and the detection line is coated with a C peptide monoclonal antibody, and the quality control line is coated with a goat anti-mouse IgG antibody. The utility model has the advantages of low background, strong fluorescent signal, high signal to noise ratio and the like, and obviously improves the sensitivity of C peptide detection. The C peptide content in human serum, plasma and whole blood is quantitatively measured in vitro, the linear range of the C peptide measured is 0.3-20ng/mL, and the lowest detection limit is 0.3ng/mL.
Description
Technical Field
The utility model relates to a C peptide rare earth fluorescent microsphere kit, a detection card and a preparation method thereof.
Background
Diabetes Mellitus (DM) is a disease of insulin secretion deficiency or insulin action disorder metabolic disturbance, and is mainly manifested by urinary sugar or hyperglycemia. Diabetes mellitus is classified into two types according to pathogenesis: type I and type II. At present, clinical screening and typing diagnosis of DM are carried out mainly by detecting serum C peptide and Insulin (INS) levels, thereby providing clinical references for selecting and determining treatment schemes. In vivo, proinsulin, a secretion product of islet beta cells, is used as a precursor substance, and is split into two parts after enzyme digestion: a molecule of C peptide; a molecule of insulin. The concentration of the C peptide in blood is far higher than that of insulin and the half-life period is longer than that of insulin, and the interference caused by insulin used for treatment can be better eliminated by measuring the concentration level of the C peptide in blood, so that the functional state of islet beta cells can be accurately reflected.
Fluorescent polymer microspheres generally refer to polymer microspheres with particle sizes between nanometers and micrometers, which are loaded with fluorescent substances and excited by external energy to generate fluorescence. The preparation method of the fluorescent microsphere and the fluorescent substance applied to the fluorescent microsphere are numerous, and are generally limited to inorganic quantum dots and organic dyes. The excitation spectrum of the inorganic quantum dot is wide, and the emission wavelength is adjustable, so that the inorganic quantum dot has fluorescence of various colors; the organic dye comprises a plurality of dyes with different excitation wavelengths, and can meet various application requirements. However, the problems of large interference of background fluorescence and stray fluorescence of the two fluorescent substances, short fluorescence lifetime and the like limit the sensitivity and accuracy of the biological detection. Meanwhile, the lanthanide rare earth nanomaterial has excellent chemical and optical properties: high luminous efficiency, long fluorescence lifetime, large Stokes shift (Stokes shift is the difference between the strongest wavelengths in the absorption spectrum and the emission spectrum of the same electron transition), narrow emission spectrum, and the like. Therefore, the rare earth complex can effectively replace inorganic quantum dots and organic dyes, thereby obtaining rare earth fluorescent microspheres, eliminating interference of background fluorescence and stray light and improving sensitivity and accuracy of biological detection.
At present, the detection means of the C peptide level mainly comprise a chemical method and an immunological method. The chemical method has the advantages of easy operation, quick detection, etc., but has poor selectivity, easy interference by external environment, strong dependence of luminous intensity on environment, easy occurrence of false positive. The immune method is based on antigen-antibody specific binding, and has high specificity and sensitivity. Therefore, the C peptide rare earth fluorescent microsphere immunochromatography detection card is prepared by adopting the rare earth fluorescent microsphere as a labeled antibody material, so that the interference of non-specific fluorescence can be effectively eliminated, and the analysis sensitivity is greatly improved.
Disclosure of Invention
The utility model aims to provide a C peptide rare earth fluorescent microsphere kit, a detection card and a preparation method thereof.
The technical scheme for realizing the aim of the utility model is as follows: the C peptide rare earth fluorescent microsphere detection card comprises a card shell and a detection test strip arranged in the card shell; the detection test strip comprises a bottom lining, a sample pad, a bonding pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the bonding pad, the nitrocellulose membrane and the absorbent paper are sequentially lapped and stuck on the bottom lining along the length direction; the binding pad is sprayed with a microsphere line formed by a C peptide monoclonal antibody marked by a C peptide rare earth fluorescent microsphere; the rare earth fluorescent microsphere is a rare earth ion complex doped fluorescent microsphere; the nitrocellulose membrane is provided with a detection line and a quality control line in parallel, wherein the detection line is close to the bonding pad, and the quality control line is far away from the bonding pad; and the detection line is coated with a C peptide monoclonal antibody, and the quality control line is coated with a goat anti-mouse IgG antibody.
The particle size of the rare earth fluorescent microsphere is 221.65 +/-9.48 nm.
The content of the C peptide monoclonal antibody marked by the C peptide rare earth fluorescent microsphere sprayed on the bonding pad is 50-200 mug antibody/200 mug fluorescent microsphere; the coating concentration of the C peptide monoclonal antibody on the detection line is 0.3-2 mg/mL.
The sample pad adopts a blood filtering membrane, preferably a whole blood filtering membrane, can realize direct detection of whole blood, can completely filter red blood cells, and is not required to be treated and directly used.
The clamping shell comprises a plastic upper shell and a plastic lower shell which are mutually clamped; the plastic upper shell is provided with a sample adding hole, an observation window and a one-dimensional code information area; the position of the sample adding hole corresponds to the position of the sample pad; the position of the observation window corresponds to the position of the nitrocellulose membrane; the one-dimensional code information area is sprayed with one-dimensional codes corresponding to detection items for the identification of the instrument.
The plastic upper shell is provided with a sample adding groove, and the groove wall of the sample adding groove is an inward concave cambered surface; the sample adding hole is arranged on the bottom surface of the sample adding groove; and a gap is arranged between the bottom surface of the sample adding groove and the detection test strip.
An anti-skid area is also arranged on the plastic upper shell; the anti-skid area is an inward concave cambered surface, and a plurality of salient points are uniformly distributed in the anti-skid area.
The utility model also provides: the C peptide rare earth fluorescent microsphere kit comprises a detection card and an ID card containing a calibration curve; the detection card adopts the C peptide rare earth fluorescent microsphere detection card.
According to the principle of a double-antibody sandwich method, the kit quantitatively detects the content of C Peptide (CP) in human serum, plasma and whole blood by utilizing an immunochromatography technology. The C Peptide (CP) in the sample is combined with a fluorescent-labeled anti-C Peptide (CP) antibody to form an immune complex. The immune complex flows upwards from capillary action to react with the anti-CP antibody in the test area, and generates a fluorescent signal in the reaction area after being excited by the excitation light source with specific wavelength. The intensity of the signal is proportional to the content of C Peptide (CP) in the sample.
The utility model also provides: the preparation method of the C peptide rare earth fluorescent microsphere kit comprises the following steps:
step one, preparing rare earth fluorescent microspheres;
step two, activating rare earth fluorescent microspheres;
preparing a rare earth fluorescent microsphere marked C peptide monoclonal antibody;
step four, preparing a sample pad and a bonding pad;
step five, treating the nitrocellulose membrane;
and step six, sequentially and mutually bonding the sample pad, the bonding pad, the nitrocellulose membrane and the absorbent paper on the substrate to obtain a test paper board, and then cutting the test paper board into test paper strips.
Step seven, assembling the test strip in a clamping shell;
and step eight, preparing an ID card containing a standard curve.
The first step is to polymerize europium complex with styrene (St) and MAA (methacrylic acid) by microemulsion polymerization, and finally to synthesize the rare earth fluorescent microsphere by one-step copolymerization. The method comprises the following specific steps: adding 50mL of ultrapure water into the conical flask, and vacuumizing for 30min to remove oxygen in the water; weighing 0.015g of emulsifier SDBS (sodium dodecyl benzene sulfonate) and placing the emulsifier SDBS into a 50mL three-neck flask, adding 23mL of ultrapure water treated by the steps, and magnetically stirring to dissolve the SDBS to obtain a water phase; respectively taking 0.1mL of MAA (methacrylic acid) and 0.9mL of styrene in a 1.5mL centrifuge tube, simultaneously weighing 0.03g of europium complex, adding the europium complex into the mixed solution, and uniformly dispersing by ultrasonic to obtain an oil phase; sucking the oil phase in the steps by using a dropper, dropwise adding the oil phase into the water phase, stirring while adding to uniformly mix, and carrying out ultrasonic fine emulsification on the oil-water mixed solution for 30min; preparing an initiator KPS (potassium persulfate) aqueous solution with a certain concentration (30 mg/mL), pouring the aqueous solution into the oil-water phase mixed solution after ultrasonic treatment, and carrying out ultrasonic treatment for 5min to uniformly mix the aqueous solution; adding the solution after ultrasonic treatment into a three-neck flask, carrying out water bath at 65 ℃ and under the protection of N2, and carrying out condensation reflux and reaction for 8 hours when the temperature rises to 70 ℃; centrifuging the reaction product at 4000rpm/min for 4min; taking the supernatant, carrying out centrifugation at 15000rpm/min for 20min, and discarding the supernatant; after the precipitate is washed once by ultrapure water, the precipitate is finally dispersed into 10mL of ultrapure water to obtain the rare earth fluorescent microspheres.
By adopting the technical scheme, the utility model has the following beneficial effects: (1) The C peptide rare earth fluorescent microsphere detection card bonding pad is sprayed with the microsphere line formed by the C peptide monoclonal antibody marked by the C peptide rare earth fluorescent microsphere, the rare earth fluorescent microsphere can be synthesized in batch, has uniform size and morphology, has excellent performances of good water solubility, easy surface modification, single particle luminous intensity, long fluorescence service life and the like, and has the advantages of low background, strong fluorescence signal, high signal to noise ratio and the like compared with imported fluorescent microsphere in the application of an in-vitro detection kit, thereby remarkably improving the sensitivity of C peptide detection. The C peptide content in human serum, plasma and whole blood is quantitatively measured in vitro, the linear range of the C peptide measured is 0.3-20ng/mL, and the lowest detection limit is 0.3ng/mL.
(2) The sample pad of the C peptide rare earth fluorescent microsphere detection card adopts a blood filtering film, preferably a whole blood filtering film, can realize direct detection of whole blood, can completely filter red blood cells, and is unnecessary to process and directly use.
(3) The clamping shell of the C peptide rare earth fluorescent microsphere detection card comprises a plastic upper shell and a plastic lower shell which are mutually clamped, and the structure enables the detection test strip to be replaced according to the requirement, and the clamping shell can be reused.
(4) The sample adding groove is formed in the upper shell of the shell plastic of the C peptide rare earth fluorescent microsphere detection card, the groove wall of the sample adding groove is an inward concave cambered surface, and the bottom surface of the sample adding groove is provided with the sample adding hole, so that samples can be prevented from being added in the sample Kong Canliu; a gap is arranged between the bottom surface of the sample adding groove and the detection test strip, so that sample overflow can be avoided, and accuracy and sensitivity can be improved.
(5) The anti-slip area is arranged on the shell plastic of the C peptide rare earth fluorescent microsphere detection card, and a plurality of salient points are uniformly distributed in the anti-slip area, so that the operation of holding the C peptide rare earth fluorescent microsphere detection card is convenient.
Drawings
In order that the utility model may be more readily understood, a more particular description of the utility model will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which
FIG. 1 is a schematic structural diagram of a C-peptide rare earth fluorescent microsphere detection card of the utility model.
FIG. 2 is a schematic diagram of the structure of the test strip of the C-peptide rare earth fluorescent microsphere detection card of the utility model.
FIG. 3 is a transmission electron microscope image of rare earth europium complex doped fluorescent microspheres in example 1 of the present utility model.
FIG. 4 shows fluorescence emission spectra and excitation spectra of rare earth europium complex-doped fluorescent microspheres in example 1 of the present utility model.
FIG. 5 is a standard graph of the C-peptide rare earth fluorescent microsphere kit of example 1 of the present utility model.
The reference numerals in the drawings are:
the sample detection device comprises a base liner 1, a sample pad 2, a bonding pad 3, a nitrocellulose membrane 4, water absorption paper 5, a detection line 6, a quality control line 7, a plastic upper shell 8, a plastic lower shell 9, a sample adding hole 10, an observation window 11, a one-dimensional code information area 12, a sample adding groove 13 and an anti-skid area 14.
Detailed Description
Example 1
The C peptide rare earth fluorescent microsphere kit comprises a detection card and an ID card containing a calibration curve; the detection card comprises a card shell and a detection test strip arranged in the card shell.
Referring to fig. 2, the test strip includes a base sheet, a sample pad 2, a bonding pad 3, a nitrocellulose membrane 4, and a bibulous paper 5 sequentially attached to the base sheet 1 in a lap joint manner in a length direction.
The binding pad 3 is sprayed with a microsphere line formed by C peptide monoclonal antibodies (the content of which is 50-200 mug antibodies/200 mug fluorescent microspheres) marked by C peptide rare earth fluorescent microspheres; the rare earth fluorescent microspheres are rare earth ion complex doped fluorescent microspheres with the particle size of 221.65 +/-9.48 nm, preferably rare earth europium elements (the solid content is 1% w/w) doped with the rare earth fluorescent microspheres and the particle size of 221.65 +/-9.48 nm; the rare earth fluorescent microsphere is stable in a ground state, and emits fluorescence with the wavelength range of 600-620 nm under the action of an excitation light source of 320-380 nm.
A detection line 6 and a quality control line 7 are arranged on the nitrocellulose membrane 4 in parallel, wherein the detection line 6 is close to the bonding pad 3, and the quality control line 7 is far away from the bonding pad 3; the distance between the detection line 6 and the quality control line 7 is 3-5 mm; the detection line 6 is coated with a C peptide monoclonal antibody, and the quality control line 7 is coated with a goat anti-mouse IgG antibody. The coating concentration of the C peptide monoclonal antibody on the detection line 6 is 0.3-2 mg/mL, the dosage is 0.5-1.5 mu L of coating liquid amount/cm membrane, and the coating concentration of the goat anti-mouse IgG antibody in the quality control line 7 is 0.3-2 mg/mL, and the dosage is 0.5-1.5 mu L of coating liquid amount/cm membrane.
The sample pad 2 adopts a blood filtering membrane, preferably a whole blood filtering membrane, can realize direct detection of whole blood, can completely filter red blood cells, and is not required to be treated and directly used.
Referring to fig. 1, the cartridge comprises a plastic upper casing 8 and a plastic lower casing 9 which are mutually clamped, and the structure enables the test strip to be replaced according to the need, and the cartridge can be reused; a sample adding hole 10, an observation window 11 and a one-dimensional code information area 12 are arranged on the plastic upper shell 8; the position of the sample adding hole 10 corresponds to the position of the sample pad 2; the position of the observation window 11 corresponds to the position of the nitrocellulose membrane 4; the one-dimensional code information area 12 is sprayed with a one-dimensional code corresponding to the detection item for identification of the instrument.
Still referring to fig. 1, a sample adding groove 13 is arranged on the plastic upper shell 8, and the groove wall of the sample adding groove 13 is an inward concave cambered surface; the sample adding hole 10 is arranged on the bottom surface of the sample adding groove 13, so that the sample can be prevented from remaining in the sample adding hole 10; a gap is arranged between the bottom surface of the sample adding groove 13 and the detection test strip, so that the overflow of the sample can be avoided, and the accuracy and the sensitivity can be improved.
The plastic upper shell 8 is also provided with an anti-skid area 14; the anti-skid area 14 is a concave cambered surface, and a plurality of salient points are uniformly distributed in the anti-skid area 14.
The preparation method of the C peptide rare earth fluorescent microsphere kit of the embodiment comprises the following steps:
step one, preparing rare earth fluorescent microspheres: adding 50mL of ultrapure water into the conical flask, and vacuumizing for 30min to remove oxygen in the water; weighing 0.015g of emulsifier SDBS (sodium dodecyl benzene sulfonate) and placing the emulsifier SDBS into a 50mL three-neck flask, adding 23mL of ultrapure water treated by the steps, and magnetically stirring to dissolve the SDBS to obtain a water phase; respectively taking 0.1mL of MAA (methacrylic acid) and 0.9mL of styrene in a 1.5mL centrifuge tube, simultaneously weighing 0.03g of europium complex, adding the europium complex into the mixed solution, and uniformly dispersing by ultrasonic to obtain an oil phase; sucking the oil phase in the steps by using a dropper, dropwise adding the oil phase into the water phase, stirring while adding to uniformly mix, and carrying out ultrasonic fine emulsification on the oil-water mixed solution for 30min; preparing an initiator KPS (potassium persulfate) aqueous solution with a certain concentration (30 mg/mL), pouring the aqueous solution into the oil-water phase mixed solution after ultrasonic treatment, and carrying out ultrasonic treatment for 5min to uniformly mix the aqueous solution; adding the solution after ultrasonic treatment into a three-neck flask, carrying out water bath at 65 ℃ and under the protection of N2, and carrying out condensation reflux and reaction for 8 hours when the temperature rises to 70 ℃; centrifuging the reaction product at 4000rpm/min for 4min; taking the supernatant, carrying out centrifugation at 15000rpm/min for 20min, and discarding the supernatant; after the precipitate is washed once by ultrapure water, the precipitate is finally dispersed into 10mL of ultrapure water to obtain the rare earth fluorescent microspheres.
The fluorescent microsphere is characterized by adopting a Transmission Electron Microscope (TEM), and the transmission electron microscope image shows the morphology and the nano-size of the rare earth fluorescent microsphere synthesized by the embodiment. As shown in FIG. 3, the rare earth fluorescent microsphere synthesized by the embodiment has good monodispersity and complete morphology. The fluorescence spectrum, as shown in FIG. 4, shows the wavelength ranges of the excitation spectrum and the emission spectrum of the fluorescent microspheres.
Step two, activating rare earth fluorescent microspheres: after ultrasonic treatment of the fluorescent microspheres for 2min, taking 200 mu L of fluorescent microspheres, centrifuging at 14000rpm for 15min, washing the precipitate to 1mL by using 100mM MES solution with pH of 6.0, and ultrasonic treatment for 2min; 50 μl of 100mg/mL carbodiimide was added, mixed well for 5min, 100 μl of 100 mg/mLN-hydroxysulfosuccinimide was added, mixed well for 15min, centrifuged at 14000rpm for 15min, and the precipitate was washed to 1mL with MES solution at pH 6.0.
Preparing a rare earth fluorescent microsphere marked C peptide monoclonal antibody: after the activated fluorescent microspheres were sonicated for 2min, C-peptide monoclonal antibodies were added at 100. Mu.g/200. Mu.L, mixed for 2 hours, blocked with 50mM, pH=8.0 Tris-HCl blocking solution containing 0.5% BSA, 0.1% glycine for 1 hour, centrifuged at 14000rpm for 15min at high speed, washed twice with buffer in Tris-HCl preservation solution containing 1% NaCl, 0.5% BSA, 0.5% sucrose, 0.1% Tween20, pH=8.0, and resuspended to 200. Mu.L at 4℃for light-shielding and storage.
Step four, preparing a sample pad 2 and a bonding pad 3: the sample pad 2 selects a whole blood filtering membrane, so that the whole blood can be directly detected, red blood cells can be completely filtered, and the whole blood filtering membrane is directly used without treatment; the rare-earth fluorescent microsphere-labeled C-peptide monoclonal antibody was diluted 15-fold with a microsphere dilution (20 mM Tris-HCl buffer containing 0.5% BSA, 25% sucrose) on the conjugate pad 3 and uniformly sprayed with a line (i.e., microsphere line) in an amount of 4. Mu.l of liquid per cm of sample pad 2. Placed in an oven and dried overnight at 37 ℃.
Step five, treating the nitrocellulose membrane 4: c peptide monoclonal antibody and goat anti-mouse IgG antibody are respectively adjusted to 0.5mg/mL and 1mg/mL by coating buffer solution (10 mM BS buffer solution containing 2.5% sucrose) with the dosage of 1 mu L of coating liquid amount/cm membrane, and are respectively used as a detection line 6 and a quality control line 7 to be parallelly marked on a nitrocellulose membrane 4 for coating, the space between the quality control line 7 and the detection line 6 is 4mm, and the mixture is dried in an oven at the humidity of less than 30% and the temperature of 37 ℃ for 10 hours, and then the mixture is sealed for standby.
Step six, a sample pad 2 (size of 17×300mm, blood filtering film material), a bonding pad 3 (16×300mm, glass fiber cotton material), a nitrocellulose membrane 4 (size of 25×300 mm) and a water absorbing paper 5 (size of 28×300 mm) are sequentially adhered to a base liner 1 (size of 80×300 mm) in a lap joint manner to obtain a test paper board, and then cut into test paper strips.
And step seven, assembling the test strip in the clamping shell.
Step eight, preparing an ID card containing a standard curve: each box contains an ID card of a standard curve (the standard curve of the same batch is the same), quality control materials with different concentrations are measured through a detection card, the concentration of the quality control materials is taken as an abscissa, the ratio of fluorescent signals is taken as an ordinate, the standard curve is drawn, the ID card is written in, one-dimensional codes are generated, and corresponding one-dimensional code information on the reagent card can be read through a dry type fluorescent immunoassay analyzer to measure the corresponding concentration.
The quantitative detection of the C peptide rare earth fluorescent microsphere kit in the embodiment is as follows:
(1) drawing of standard curve
The time-resolved fluorescence detection card prepared in example 1 was added with different concentrations of C peptide antigen quality control substances (7 concentrations of 0.3, 1, 2, 4, 8, 15 and 20ng/mL, three replicates were set for each concentration), C peptide antigen was diluted with calf serum to obtain 20ng/L quality control substance mother liquor, and then diluted with calf serum to different quality control substance concentrations), after chromatography for 15min, C, T line fluorescence signals and C/T values were read by an AFS-1000 type dry fluorescence immunoassay instrument manufactured by Lanbo biotechnology Co., guangzhou, and experimental results and analyses are shown in Table 1:
and drawing a standard curve by using the concentration of the C peptide quality control product and the average value of the sample signal T/C, wherein curve data are shown in table 1, and the standard curve is shown in fig. 5. Wherein the R value is 0.9974, and the concentration of the C peptide contained in the sample is quantitatively determined by the marked line.
(2) Performance test of time-resolved fluorescence test paper card
Minimum detection limit: the measurement is repeated for 20 times by using zero value samples, the average value M and the standard deviation ID of the 20 times results are calculated, the detection limit (M+2 ID) of the standard deviation reporting method is added by twice to the blank average value, the result is 0.24ng/mL, and the sensitivity is 0.3ng/mL.
Linear range: taking 7 concentration values between 0.2 and 10ng/mL, repeatedly measuring each concentration for three times, and carrying out linear analysis on the average value of the measured concentration and the theoretical concentration to obtain a linear equation y=0.996x+0.0912 and r=0.9995, wherein the correlation of the kit is good in the linear range of 0.3 to 20 ng/mL.
Precision: three batches of the C peptide rare earth fluorescent microsphere kits of the embodiment are respectively detected for 15ng/mL and 4ng/mL of repetitive property control products, each batch of the kit is parallelly detected for 10 times by using the repetitive property control products, the CV (constant velocity) of the three batches with the concentration of 15ng/mL is respectively 7.01%, 5.72% and 6.14%, the CV (constant velocity) of the three batches is 6.21%, the CV (constant velocity) of the three batches with the concentration of 4ng/mL is respectively 6.86%, 8.77% and 7.81%, and the CV (constant velocity) of the three batches is 7.63% and is within 10%.
Accuracy: selecting a quality control material with the concentration of 1ng/mL as a detection sample, dividing the detection sample into 3 parts with the same volume, respectively adding 15ng/mL and 4ng/mL of accuracy quality control materials into 2 parts of samples, preparing 2 recovered samples with different added concentrations, and calculating the concentration of the added object to be detected. The same amount of solution without the test object is added into another sample to prepare a basic sample. The sample was analyzed 3 times repeatedly and the mean value was calculated. Calculate recovery = (analysis sample concentration-base sample concentration)/addition concentration x 100%. The recovery rate of 15ng/mL of the recovered sample was 97.78%, the recovery rate of 4ng/mL was 95.53%, and the average recovery rate was 96.56%. The deviation is within 10%.
While the foregoing is directed to embodiments of the present utility model, other and further details of the utility model may be had by the present utility model, it should be understood that the foregoing description is merely illustrative of the present utility model and that no limitations are intended to the scope of the utility model, except insofar as modifications, equivalents, improvements or modifications are within the spirit and principles of the utility model.
Claims (5)
- C peptide rare earth fluorescent microsphere detection card, its characterized in that: comprises a card shell and a detection test strip arranged in the card shell; the detection test strip comprises a bottom lining, a sample pad, a bonding pad, a nitrocellulose membrane and absorbent paper, wherein the sample pad, the bonding pad, the nitrocellulose membrane and the absorbent paper are sequentially lapped and stuck on the bottom lining along the length direction; the binding pad is sprayed with microsphere lines formed by C peptide monoclonal antibodies marked by rare earth fluorescent microspheres; the rare earth fluorescent microsphere is a rare earth ion complex doped fluorescent microsphere; the nitrocellulose membrane is provided with a detection line and a quality control line in parallel, wherein the detection line is close to the bonding pad, and the quality control line is far away from the bonding pad; the detection line is coated with a C peptide monoclonal antibody, and the quality control line is coated with a goat anti-mouse IgG antibody;the clamping shell comprises a plastic upper shell and a plastic lower shell which are mutually clamped; the plastic upper shell is provided with a sample adding hole, an observation window and a one-dimensional code information area; the position of the sample adding hole corresponds to the position of the sample pad; the position of the observation window corresponds to the position of the nitrocellulose membrane; the one-dimensional code information area is sprayed with one-dimensional codes which are identified by the instrument and correspond to the detection items;the plastic upper shell is provided with a sample adding groove, and the groove wall of the sample adding groove is an inward concave cambered surface; the sample adding hole is arranged on the bottom surface of the sample adding groove; a gap is arranged between the bottom surface of the sample adding groove and the detection test strip;an anti-skid area is also arranged on the plastic upper shell; the anti-skid area is an inward concave cambered surface, and a plurality of salient points are uniformly distributed in the anti-skid area;the preparation method of the rare earth fluorescent microsphere comprises the following steps: adding 50mL of ultrapure water into the conical flask, and vacuumizing for 30min to remove oxygen in the water; weighing 0.015g of emulsifier sodium dodecyl benzene sulfonate, placing the emulsifier sodium dodecyl benzene sulfonate into a 50mL three-necked flask, adding 23mL of ultrapure water treated by the steps, and magnetically stirring to dissolve the sodium dodecyl benzene sulfonate to obtain a water phase; respectively taking 0.1mL of methacrylic acid and 0.9mL of styrene in a 1.5mL centrifuge tube, simultaneously weighing 0.03g of europium complex, adding the europium complex into the mixed solution, and uniformly dispersing by ultrasonic to obtain an oil phase; sucking the oil phase in the steps by using a dropper, dropwise adding the oil phase into the water phase, stirring while adding to uniformly mix, and carrying out ultrasonic fine emulsification on the oil-water mixed solution for 30min; preparing 30mg/mL of initiator potassium persulfate aqueous solution, pouring the solution into the oil-water phase mixed solution after ultrasonic treatment, and carrying out ultrasonic treatment for 5min to uniformly mix the solution; adding the ultrasonic solution into a three-neck flask, carrying out water bath at 65 ℃ and N 2 Under the protection condition, when the temperature is raised to 70 ℃, condensing and refluxing are carried out, and the reaction is carried out for 8 hours; centrifuging the reaction product at 4000rpm/min for 4min; taking the supernatant, carrying out centrifugation at 15000rpm/min for 20min, and discarding the supernatant; washing the precipitate with ultrapure water once, and finally dispersing the precipitate into 10mL of ultrapure water to obtain rare earth fluorescent microspheres; the particle size of the rare earth fluorescent microsphere is 221.65 +/-9.48 nm.
- 2. The C-peptide rare earth fluorescent microsphere assay card of claim 1, wherein: the content of the rare earth fluorescent microsphere marked C peptide monoclonal antibody sprayed on the bonding pad is 50-200 mug antibody/200 mug fluorescent microsphere; the coating concentration of the C peptide monoclonal antibody on the detection line is 0.3-2 mg/mL.
- 3. The C-peptide rare earth fluorescent microsphere assay card of claim 1, wherein: the sample pad adopts a blood filtering membrane.
- 4. A C peptide rare earth fluorescent microsphere kit is characterized in that: comprises a detection card and an ID card containing a calibration curve; the detection card adopts the C peptide rare earth fluorescent microsphere detection card according to any one of claims 1 to 3.
- 5. The method for preparing the C peptide rare earth fluorescent microsphere kit according to claim 4, comprising the following steps:step one, preparing rare earth fluorescent microspheres;step two, activating rare earth fluorescent microspheres;preparing a rare earth fluorescent microsphere marked C peptide monoclonal antibody;step four, preparing a sample pad and a bonding pad;step five, treating the nitrocellulose membrane;step six, sequentially and mutually bonding a sample pad, a bonding pad, a nitrocellulose membrane and absorbent paper on the substrate to obtain a test paper board, and then cutting the test paper board into test paper strips;step seven, assembling the test strip in a clamping shell;and step eight, preparing an ID card containing a standard curve.
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