CN113848326A - Urine microalbumin detection kit and preparation thereof - Google Patents

Urine microalbumin detection kit and preparation thereof Download PDF

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Publication number
CN113848326A
CN113848326A CN202111053687.1A CN202111053687A CN113848326A CN 113848326 A CN113848326 A CN 113848326A CN 202111053687 A CN202111053687 A CN 202111053687A CN 113848326 A CN113848326 A CN 113848326A
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China
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urine microalbumin
product
urine
detection
antibody
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王胜岚
涂妮娜
袁瑜容
彭永林
肖慧芳
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Pinet Zhongshan Biotechnology Co ltd
Zhongshan Institute of Modern Industrial Technology of South China University of Technology
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Pinet Zhongshan Biotechnology Co ltd
Zhongshan Institute of Modern Industrial Technology of South China University of Technology
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Priority to CN202111053687.1A priority Critical patent/CN113848326A/en
Publication of CN113848326A publication Critical patent/CN113848326A/en
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Abstract

The invention relates to a urine microalbumin detection kit and preparation thereof. The kit comprises a urine microalbumin detection card, a urine microalbumin diluent and a urine microalbumin chip; the urine microalbumin detection card comprises a card shell and a test strip; the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC base plate, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially stuck on the PVC base plate; the combination pad contains a fluorescence-labeled urine microalbumin antibody and a fluorescence-labeled goat anti-chicken IgY antibody. The kit can accurately and quantitatively detect the content of microalbumin in human urine, and the labeled europium fluorescent nano-microspheres are connected with the antibody through chemical bonds, so that the labeled product is stable. Has the characteristics of excellent water solubility, high sensitivity, accurate quantification, simplicity, convenience and quickness.

Description

Urine microalbumin detection kit and preparation thereof
Technical Field
The invention relates to a urine microalbumin detection kit and a preparation method thereof.
Background
Urine microalbumin (mALB) refers to the presence of microalbumin in urine. Albumin is a normal protein in blood, and under the condition of normal metabolism of human body, the albumin in urine is very little, and every liter of urine albumin is not more than 20mg, so that the albumin is called microalbumin. However, early-stage kidney damage such as diabetic nephropathy and hypertensive nephropathy and complications of cardiovascular diseases cause kidney-inherent cell damage, and the structure and function of kidney-inherent cells are changed, so that the albumin content is increased in urine. Urine microalbumin is found in diabetic nephropathy, hypertension and preeclampsia and is an early sensitive index of kidney injury.
The prior art method for measuring microalbumin in urine is mainly an immunoassay, i.e. an analysis method for qualitatively and quantitatively measuring an antigen or an antibody by using the characteristic reaction of the antigen and the antibody. The immunoassay method can be mainly classified into: radioimmunoassay, enzyme immunoassay, luminescence immunoassay, and fluorescence immunoassay. The fluoroimmunoassay refers to a method of labeling an antibody or an antigen with a fluorescent molecule and then observing the labeled fluorescence with a fluorescence microscope to analyze tracing the corresponding antigen or antibody. Compared to the other three immunization methods, the fluorescence immunization method has many advantages: the selectivity is good; secondly, various measurement parameters (such as fluorescence anisotropy, fluorescence lifetime, fluorescence quantum yield, fluorescence excitation wavelength, emission wavelength and the like) are provided; high sensitivity; and fourthly, the method has a plurality of detection techniques and methods (such as derivative fluorescence, synchronous fluorescence, fluorescence polarization, fluorescence kinetic analysis method, time-resolved fluorescence analysis method and the like). Therefore, the method can be selected according to different actual measurement conditions, has the characteristics of strong applicability, good selectivity and wider linear range, and is a simple and practical analysis technology.
The most commonly used labels for fluoroimmunoassay are organic dyes. However, organic dyes have many defects, such as photobleaching phenomenon of most fluorescent reagents, which results in unstable fluorescence signals; the fluorescent reagent has certain toxic and side effects; the excitation spectrum of the fluorescent dye is narrow, so that multiple components are difficult to excite simultaneously, the fluorescence spectrum is wide, the distribution is asymmetric, the fluorescence of different probe molecules is difficult to distinguish, and the multiple components cannot be detected simultaneously; in addition, the traditional fluorescence labeling method can only connect a few fluorescent molecules on the active groups of the biomolecules, and the analysis sensitivity is low. Therefore, the research on novel fluorescent probes with high sensitivity and good stability is a hotspot of the current fluorescent immunoassay field.
Disclosure of Invention
The invention aims to provide a carboxyl modified europium nano-microsphere fluorescence immunochromatographic technique aiming at the defects that the traditional fluorescence labeling method in the prior art can only connect a few fluorescent molecules on active groups of biomolecules and has lower analysis sensitivity, and a kit and a method for quantitatively detecting trace urine albumin, which have high sensitivity, can accurately quantify, have excellent water solubility and are simple, convenient and quick.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a urine microalbumin detection kit comprises a urine microalbumin detection card, a urine microalbumin diluent and a urine microalbumin chip:
the urine microalbumin detection card comprises a card shell and a test strip;
the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC base plate, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially stuck on the PVC base plate;
the combination pad contains a urine microalbumin antibody which is fluorescently labeled and a goat anti-chicken IgY antibody which is fluorescently labeled;
the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, the detection line and the quality control line are mutually parallel, the distance is 3-5mm, and the detection line is close to the sample adding hole; the detection line is coated with a urine microalbumin monoclonal antibody, and the quality control line is coated with a chicken IgY antibody.
Preferably, the preparation method of the test strip is as follows:
(1) preparation of fluorescent nano-microspheres
Adding 50ml of absolute ethyl alcohol, 3ml of ammonia water and 1ml of water into a three-neck flask in sequence, heating to 40 ℃, keeping the temperature, stirring, quickly adding 1.5ml of tetraethoxysilane, reacting for 7 hours, then adding 1ml of tetraethoxysilane, and continuing to react for 15-20 hours; centrifuging at high speed, washing with ethanol and water for three times respectively, and redissolving in water to obtain silicon dioxide nanoparticles which are marked as a product I;
weighing 1mmol Eu2O3Placing in a beaker, adding 10ml 20% diluted hydrochloric acid, stirring for dissolving, heating at 80-100 deg.C for drying, removing excessive HCl, adding 20ml anhydrous ethanol for dissolving to obtain 2mmol EuCl3The absolute ethanol solution is marked as a product II;
weighing 4mmo1 benzoylacetone, dissolving in 20ml ethanol, slowly dripping the product II into the ethanol solution of the benzoylacetone under stirring at 60 ℃, adjusting the pH to 6-7 by NaOH, and generating a large amount of precipitate; heating, refluxing and stirring for 30min, then dropwise adding 10ml of an ethanol solution containing 2mmol of phenanthroline, continuously stirring for 1h, and drying to obtain a product III;
weighing 1mmol of salicylic acid and 1mmol of triethylamine, dissolving in 15ml of dichloromethane, dissolving 1mmol of acryloyl chloride in 3ml of dichloromethane, placing in a constant pressure dropping funnel, controlling the dropping speed, carrying out ice bath, completing dropping within 1 hour, and stirring for 6-12 hours; washing twice with 20% dilute hydrochloric acid, washing twice with saturated NaOH solution, washing three times with deionized water, separating liquid, taking an organic phase, drying, and distilling under reduced pressure to obtain a product IV;
weighing 25mg of product IV and 3.5-11mg of acrylic acid, dissolving in 5ml of dichloromethane, stirring, mixing uniformly, adding 0.5mg of AIBN, N2Protecting, reacting at 65 ℃ for 24 hours, adding 50ml of absolute methanol into the reaction solution to generate white precipitate, washing with absolute ethanol twice, and washing with deionized water three times to obtain a product V;
dissolving the product III in 30ml of DMF, weighing 2mmol of the product V, dissolving the product V in 10ml of DMF, adjusting the pH to 6-7 by using 1M of NaOH aqueous solution, stirring the solution in a water bath kettle at the temperature of 50-60 ℃ for 24-48h, allowing the solution to stand for 24h, performing suction filtration, separating out a precipitate, filtering, repeatedly washing the precipitate by using a small amount of ethanol, and drying to obtain fluorescent nano microspheres, namely the target product;
(2) activation of fluorescent nanospheres
Putting 100 mul of fluorescent nano-microspheres into a 1.5ml EP tube, adding 100 mul of 1mg/ml EDC and 100 mul of 0.6mg/ml NHS, activating for 60min at 37 ℃, centrifuging at high speed of 14000rpm, removing the rest EDC and NHS, and washing with water for three times to obtain a target product;
(3) preparation of fluorescence-labeled urine microalbumin antibody
Incubating 100 μ l of 20 μ g/ml urine microalbumin antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark place to obtain a target product;
(4) preparation of fluorescent-labeled goat anti-chicken IgY antibody
Incubating 100 μ l of 20 μ g/ml goat anti-chicken IgY antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal the residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark to obtain a target product;
(5) preparation of nitrocellulose membranes
Respectively using buffer solutions to respectively draw the urine microalbumin monoclonal antibody and the chicken IgY antibody as a detection line and a quality control line in parallel on a nitrocellulose membrane for coating, wherein the interval between the quality control line and the detection line is 3-5mm, placing the test lines in a drying oven, and drying the test lines at 45 ℃ overnight to obtain a target product;
(6) and sequentially and mutually bonding a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on the PVC base plate in a lap joint manner to obtain a test paper plate, and cutting to obtain the detection test paper strip.
Preferably, the test strip is fixed on the card shell, the surface of the test strip is pressed tightly by the card surface, and the card surface is respectively reserved with a sample adding hole and an observation window at the part corresponding to the sample pad, the combination pad and the nitrocellulose membrane.
Preferably, the urine microalbumin diluent is a phosphate buffer.
Preferably, the urine microalbumin chip is the same batch of reagent standard curve information and is not interchangeable for use.
The invention relates to a method for detecting urine microalbumin by using the kit, wherein the test is carried out at room temperature of 18-30 ℃, and the method comprises the following steps:
(1) opening the instrument and inserting a chip with the same reagent batch number;
(2) sucking 10 mu L of sample by using a pipette, adding the sample into the diluent, and turning upside down and uniformly mixing;
(3) opening the aluminum foil bag, taking out the detection card, and horizontally placing on a desktop;
(4) sucking 100 mu L of the diluted and uniformly mixed sample by using a pipettor, and adding the sample into a sample adding hole of the detection card;
(5) selecting a sample type 'urine' on a matched instrument;
(6) and (3) immediate test: after the room temperature reaction is carried out for 10min, the detection card is placed in an instrument card slot, an 'instant test' mode is selected, and a 'test' is clicked; standard test: placing the detection card into a card slot of an instrument, selecting a standard test mode, clicking a test mode, automatically timing by the instrument, and automatically testing and displaying a result after timing is finished;
(7) clicking 'printing' can print a detection result report.
The invention has the beneficial effects that:
(1) the invention provides a urine microalbumin detection kit, wherein a detection test strip adopts a carboxyl modified europium nano microsphere fluorescence immunochromatography technology, and has long fluorescence life which is usually more than 1 ms; the Stokes displacement is large, the exciting light is 365nm, the emitting light is 615nm, and the interference of scattered light caused by the exciting light on measurement can be overcome; the excitation luminescence spectrum is wider, the emission spectrum is very narrow, the fluorescence background can be further reduced, and the resolution ratio is high. Finally, the sensitivity and the specificity of the reagent are improved, and the performance of the product is improved.
(2) The invention provides a urine microalbumin detection kit, which firstly adopts a synthesized high-quality water-soluble quantum dot series product as a fluorescent labeling material to be introduced into an immunochromatography system to carry out quantitative analysis on a target object, and has the advantages of strong fluorescence intensity, good stability, good biocompatibility and the like. Secondly, detection and data analysis are carried out based on a fluorescence immunoassay POCT platform, and the method has the characteristics of multi-channel/multi-connected card detection, information code scanning, automatic card losing, automatic curve fitting, high testing speed and the like. And the turnover is fast, sample processing is not needed, detection is carried out anytime and anywhere, the operation requirement is low, and the medical cost is low. Thirdly, realizing the quantitative detection of the urine Microalbumin (MAU), the accuracy is in the range of 85-115%, the linearity is in the range of [5,200] mug/mL, and the correlation coefficient (r) is not lower than 0.990; the Coefficient of Variation (CV) in batch is not higher than 10.0 percent, and the blank limit is not more than 2.5 mu g/mL.
The specific implementation mode is as follows:
the present invention will be described in detail with reference to examples. It is to be understood, however, that the following examples are illustrative of embodiments of the present invention and are not to be construed as limiting the scope of the invention.
Example 1
A urine microalbumin detection kit comprises a urine microalbumin detection card, a urine microalbumin diluent and a urine microalbumin chip:
the urine microalbumin detection card comprises a card shell and a test strip;
the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC base plate, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially stuck on the PVC base plate;
the combination pad contains a urine microalbumin antibody which is marked by fluorescence and a goat anti-chicken IgY antibody which is marked by fluorescence;
the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, the detection line and the quality control line are mutually parallel, the distance is 3-5mm, and the detection line is close to the sample adding hole; the detection line is coated with a urine microalbumin monoclonal antibody, and the quality control line is coated with a chicken IgY antibody.
The preparation method of the carboxyl modified europium nano-microsphere fluorescence detection test strip comprises the following steps:
(1) preparation of fluorescent nano-microspheres
Adding 50ml of absolute ethyl alcohol, 3ml of ammonia water and 1ml of water into a three-neck flask in sequence, heating to 40 ℃, keeping the temperature, stirring, quickly adding 1.5ml of tetraethoxysilane, reacting for 7 hours, then adding 1ml of tetraethoxysilane, and continuing to react for 15 hours; centrifuging at high speed, washing with ethanol and water for three times respectively, and redissolving in water to obtain silicon dioxide nanoparticles which are marked as a product I;
weighing 1mmol Eu2O3Placing in a beaker, adding 10ml 20% diluted hydrochloric acid, stirring for dissolving, heating at 80 deg.C for nearly drying, removing excessive HCl, adding 20ml anhydrous ethanol for dissolving to obtain 2mmol EuCl3The absolute ethanol solution is marked as a product II;
4 mmol 1 of benzoylacetone are weighed out and dissolved in 20ml of ethanol, the product II is slowly added dropwise to an ethanol solution of benzoylacetone with stirring at 60 ℃ and the pH is adjusted to 6 with NaOH, a large amount of precipitate appears. Heating, refluxing and stirring for 30min, then dropwise adding 10ml of an ethanol solution containing 2mmol of phenanthroline, continuously stirring for 1h, and drying to obtain a product III;
weighing 1mmol of salicylic acid and 1mmol of triethylamine, dissolving in 15ml of dichloromethane, dissolving 1mmol of acryloyl chloride in 3ml of dichloromethane, placing in a constant pressure dropping funnel, controlling the dropping speed, carrying out ice bath, completing dropping within 1 hour, and stirring for 6 hours; washing twice with 20% dilute hydrochloric acid, washing twice with saturated NaOH solution, washing three times with deionized water, separating liquid, taking an organic phase, drying, and distilling under reduced pressure to obtain a product IV;
weighing 25mg of product IV,Dissolving 7.5mg acrylic acid in 5ml dichloromethane, stirring, mixing, adding 0.5mg AIBN, N2Protecting, reacting at 65 ℃ for 24 hours, adding 50ml of absolute methanol into the reaction solution to generate white precipitate, washing with absolute ethanol twice, and washing with deionized water three times to obtain a product V;
dissolving the product III in 30ml of DMF, weighing 2mmol of the product V, dissolving the product V in 10ml of DMF, adjusting the pH to 7 by using 1M of NaOH aqueous solution, stirring the solution in a water bath kettle at 50 ℃ for 48 hours, generating a precipitate, standing the solution for 24 hours, performing suction filtration, separating out the precipitate, filtering the precipitate, repeatedly washing the precipitate by using a small amount of ethanol, and drying the precipitate to obtain fluorescent nano microspheres, namely the target product;
(2) activation of fluorescent nanospheres
Putting 100 mul of fluorescent nano-microspheres into a 1.5ml EP tube, adding 100 mul of 1mg/ml EDC and 100 mul of 0.6mg/ml NHS, activating for 60min at 37 ℃, centrifuging at high speed of 14000rpm, removing the rest EDC and NHS, and washing with water for three times to obtain a target product;
(3) preparation of fluorescence-labeled urine microalbumin antibody
Incubating 100 μ l of 20 μ g/ml urine microalbumin antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark place to obtain a target product;
(4) preparation of fluorescent-labeled goat anti-chicken IgY antibody
Incubating 100 μ l of 20 μ g/ml goat anti-chicken IgY antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal the residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark to obtain a target product;
(5) preparation of nitrocellulose membranes
Respectively using buffer solutions to respectively draw the urine microalbumin monoclonal antibody and the chicken IgY antibody as a detection line and a quality control line in parallel on a nitrocellulose membrane for coating, wherein the interval between the quality control line and the detection line is 3-5mm, placing the test lines in a drying oven, and drying the test lines at 45 ℃ overnight to obtain a target product;
(6) and sequentially and mutually bonding a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on the PVC base plate in a lap joint manner to obtain a test paper plate, and cutting to obtain the detection test paper strip.
The detection test strip is fixed on the card shell, the surface of the test strip is compressed by a card surface, and the card surface is respectively reserved with a sample adding hole and an observation window at the part corresponding to the sample pad, the combination pad and the nitrocellulose membrane.
The urine microalbumin diluent is phosphate buffer.
The urine microalbumin chip is the same batch of reagent standard curve information and can not be used interchangeably.
Example 2
A urine microalbumin detection kit comprises a urine microalbumin detection card, a urine microalbumin diluent and a urine microalbumin chip:
the urine microalbumin detection card comprises a card shell and a test strip;
the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC base plate, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially stuck on the PVC base plate;
the combination pad contains a urine microalbumin antibody which is marked by fluorescence and a goat anti-chicken IgY antibody which is marked by fluorescence;
the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, the detection line and the quality control line are mutually parallel, the distance is 3-5mm, and the detection line is close to the sample adding hole; the detection line is coated with a urine microalbumin monoclonal antibody, and the quality control line is coated with a chicken IgY antibody.
The preparation method of the carboxyl modified europium nano-microsphere fluorescence detection test strip comprises the following steps:
(1) preparation of fluorescent nano-microspheres
Adding 50ml of absolute ethyl alcohol, 3ml of ammonia water and 1ml of water into a three-neck flask in sequence, heating to 40 ℃, keeping the temperature, stirring, quickly adding 1.5ml of tetraethoxysilane, reacting for 7 hours, then adding 1ml of tetraethoxysilane, and continuing to react for 20 hours; centrifuging at high speed, washing with ethanol and water for three times respectively, and redissolving in water to obtain silicon dioxide nanoparticles which are marked as a product I;
weighing 1mmol Eu2O3Placing in a furnaceAdding 10ml 20% diluted hydrochloric acid into the cup, stirring to dissolve, heating at 100 deg.C to near dry, removing excessive HCl, adding 20ml anhydrous ethanol to dissolve to obtain 2mmol EuCl3The absolute ethanol solution is marked as a product II;
4 mmol 1 of benzoylacetone are weighed out and dissolved in 20ml of ethanol, the product II is slowly added dropwise to an ethanol solution of benzoylacetone with stirring at 60 ℃ and the pH is adjusted to 7 with NaOH, a large amount of precipitate appears. Heating, refluxing and stirring for 30min, then dropwise adding 10ml of an ethanol solution containing 2mmol of phenanthroline, continuously stirring for 1h, and drying to obtain a product III;
weighing 1mmol of salicylic acid and 1mmol of triethylamine, dissolving in 15ml of dichloromethane, dissolving 1mmol of acryloyl chloride in 3ml of dichloromethane, placing in a constant pressure dropping funnel, controlling the dropping speed, carrying out ice bath, completing dropping within 1 hour, and stirring for 12 hours; washing twice with 20% dilute hydrochloric acid, washing twice with saturated NaOH solution, washing three times with deionized water, separating liquid, taking an organic phase, drying, and distilling under reduced pressure to obtain a product IV;
25mg of product IV and 3.5mg of acrylic acid are weighed out and dissolved in 5ml of dichloromethane, stirred and mixed uniformly, 0.5mg of AIBN, N are added2Protecting, reacting at 65 ℃ for 24 hours, adding 50ml of absolute methanol into the reaction solution to generate white precipitate, washing with absolute ethanol twice, and washing with deionized water three times to obtain a product V;
dissolving the product III in 30ml of DMF, weighing 2mmol of the product V, dissolving the product V in 10ml of DMF, adjusting the pH to 6 by using 1M of NaOH aqueous solution, stirring the solution in a water bath kettle at the temperature of 60 ℃ for 24 hours, generating a precipitate, standing the solution for 24 hours, carrying out suction filtration, separating out the precipitate, filtering the precipitate, repeatedly washing the precipitate by using a small amount of ethanol, and drying the precipitate to obtain fluorescent nano microspheres, namely the target product;
(2) activation of fluorescent nanospheres
Putting 100 mul of fluorescent nano-microspheres into a 1.5ml EP tube, adding 100 mul of 1mg/ml EDC and 100 mul of 0.6mg/ml NHS, activating for 60min at 37 ℃, centrifuging at high speed of 14000rpm, removing the rest EDC and NHS, and washing with water for three times to obtain a target product;
(3) preparation of fluorescence-labeled urine microalbumin antibody
Incubating 100 μ l of 20 μ g/ml urine microalbumin antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark place to obtain a target product;
(4) preparation of fluorescent-labeled goat anti-chicken IgY antibody
Incubating 100 μ l of 20 μ g/ml goat anti-chicken IgY antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal the residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark to obtain a target product;
(5) preparation of nitrocellulose membranes
Respectively using buffer solutions to respectively draw the urine microalbumin monoclonal antibody and the chicken IgY antibody as a detection line and a quality control line in parallel on a nitrocellulose membrane for coating, wherein the interval between the quality control line and the detection line is 3-5mm, placing the test lines in a drying oven, and drying the test lines at 45 ℃ overnight to obtain a target product;
(6) and sequentially and mutually bonding a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on the PVC base plate in a lap joint manner to obtain a test paper plate, and cutting to obtain the detection test paper strip.
The detection test strip is fixed on the card shell, the surface of the test strip is compressed by a card surface, and the card surface is respectively reserved with a sample adding hole and an observation window at the part corresponding to the sample pad, the combination pad and the nitrocellulose membrane.
The urine microalbumin diluent is phosphate buffer.
The urine microalbumin chip is the same batch of reagent standard curve information and can not be used interchangeably.
Example 3
A urine microalbumin detection kit comprises a urine microalbumin detection card, a urine microalbumin diluent and a urine microalbumin chip:
the urine microalbumin detection card comprises a card shell and a test strip;
the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC base plate, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially stuck on the PVC base plate;
the combination pad contains a urine microalbumin antibody which is marked by fluorescence and a goat anti-chicken IgY antibody which is marked by fluorescence;
the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, the detection line and the quality control line are mutually parallel, the distance is 3-5mm, and the detection line is close to the sample adding hole; the detection line is coated with a urine microalbumin monoclonal antibody, and the quality control line is coated with a chicken IgY antibody.
The preparation method of the carboxyl modified europium nano-microsphere fluorescence detection test strip comprises the following steps:
(1) preparation of fluorescent nano-microspheres
Adding 50ml of absolute ethyl alcohol, 3ml of ammonia water and 1ml of water into a three-neck flask in sequence, heating to 40 ℃, keeping the temperature, stirring, quickly adding 1.5ml of tetraethoxysilane, reacting for 7 hours, then adding 1ml of tetraethoxysilane, and continuing to react for 18 hours; centrifuging at high speed, washing with ethanol and water for three times respectively, and redissolving in water to obtain silicon dioxide nanoparticles which are marked as a product I;
weighing 1mmol Eu2O3Placing in a beaker, adding 10ml 20% diluted hydrochloric acid, stirring for dissolving, heating at 90 deg.C for nearly drying, removing excessive HCl, adding 20ml anhydrous ethanol for dissolving to obtain 2mmol EuCl3The absolute ethanol solution is marked as a product II;
4 mmol 1 of benzoylacetone are weighed out and dissolved in 20ml of ethanol, the product II is slowly added dropwise to an ethanol solution of benzoylacetone with stirring at 60 ℃ and the pH is adjusted to 7 with NaOH, a large amount of precipitate appears. Heating, refluxing and stirring for 30min, then dropwise adding 10ml of an ethanol solution containing 2mmol of phenanthroline, continuously stirring for 1h, and drying to obtain a product III;
weighing 1mmol of salicylic acid and 1mmol of triethylamine, dissolving in 15ml of dichloromethane, dissolving 1mmol of acryloyl chloride in 3ml of dichloromethane, placing in a constant pressure dropping funnel, controlling the dropping speed, carrying out ice bath, completing dropping within 1 hour, and stirring for 10 hours; washing twice with 20% dilute hydrochloric acid, washing twice with saturated NaOH solution, washing three times with deionized water, separating liquid, taking an organic phase, drying, and distilling under reduced pressure to obtain a product IV;
25mg of product IV and 11mg of acrylic acid are weighed out and dissolved in 5ml of dichloromethane, stirred and mixed uniformly, 0.5mg of AIBN, N are added2Protecting, reacting at 65 ℃ for 24h, adding 50ml of anhydrous methanol into the reaction solution,generating white precipitate, washing twice with absolute ethyl alcohol and washing three times with deionized water to obtain a product V;
dissolving the product III in 30ml of DMF, weighing 2mmol of the product V, dissolving the product V in 10ml of DMF, adjusting the pH to 6 by using 1M of NaOH aqueous solution, stirring the solution in a water bath kettle at 50 ℃ for 30 hours, generating a precipitate, standing the solution for 24 hours, performing suction filtration, separating out the precipitate, filtering the precipitate, repeatedly washing the precipitate by using a small amount of ethanol, and drying the precipitate to obtain fluorescent nano microspheres, namely the target product;
(2) activation of fluorescent nanospheres
Putting 100 mul of fluorescent nano-microspheres into a 1.5ml EP tube, adding 100 mul of 1mg/ml EDC and 100 mul of 0.6mg/ml NHS, activating for 60min at 37 ℃, centrifuging at high speed of 14000rpm, removing the rest EDC and NHS, and washing with water for three times to obtain a target product;
(3) preparation of fluorescence-labeled urine microalbumin antibody
Incubating 100 μ l of 20 μ g/ml urine microalbumin antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark place to obtain a target product;
(4) preparation of fluorescent-labeled goat anti-chicken IgY antibody
Incubating 100 μ l of 20 μ g/ml goat anti-chicken IgY antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal the residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark to obtain a target product;
(5) preparation of nitrocellulose membranes
Respectively using buffer solutions to respectively draw the urine microalbumin monoclonal antibody and the chicken IgY antibody as a detection line and a quality control line in parallel on a nitrocellulose membrane for coating, wherein the interval between the quality control line and the detection line is 3-5mm, placing the test lines in a drying oven, and drying the test lines at 45 ℃ overnight to obtain a target product;
(6) and sequentially and mutually bonding a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on the PVC base plate in a lap joint manner to obtain a test paper plate, and cutting to obtain the detection test paper strip.
The detection test strip is fixed on the card shell, the surface of the test strip is compressed by a card surface, and the card surface is respectively reserved with a sample adding hole and an observation window at the part corresponding to the sample pad, the combination pad and the nitrocellulose membrane.
The urine microalbumin diluent is phosphate buffer.
The urine microalbumin chip is the same batch of reagent standard curve information and can not be used interchangeably.
The following comparative examples were tested against example 1:
comparative example 1
A urine microalbumin detection kit comprises a urine microalbumin detection card, a urine microalbumin diluent and a urine microalbumin chip:
the urine microalbumin detection card comprises a card shell and a test strip;
the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC base plate, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially stuck on the PVC base plate;
the combination pad contains a urine microalbumin antibody which is marked by fluorescence and a goat anti-chicken IgY antibody which is marked by fluorescence;
the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, the detection line and the quality control line are mutually parallel, the distance is 3-5mm, and the detection line is close to the sample adding hole; the detection line is coated with a urine microalbumin monoclonal antibody, and the quality control line is coated with a chicken IgY antibody.
The preparation method of the carboxyl modified europium nano-microsphere fluorescence detection test strip comprises the following steps:
(1) preparation of fluorescent nano-microspheres
Adding 50ml of absolute ethyl alcohol, 3ml of ammonia water and 1ml of water into a three-neck flask in sequence, heating to 40 ℃, keeping the temperature, stirring, quickly adding 1.5ml of tetraethoxysilane, reacting for 7 hours, then adding 1ml of tetraethoxysilane, and continuing to react for 15 hours; centrifuging at high speed, washing with ethanol and water for three times respectively, and redissolving in water to obtain silicon dioxide nanoparticles which are marked as a product I;
weighing 1mmol Eu2O3Placing in a beaker, adding 10ml 20% diluted hydrochloric acid, stirring for dissolving, heating at 80 deg.C for nearly drying, and removingAdding HCl, adding 20ml absolute ethyl alcohol to dissolve to obtain 2mmol EuCl3The absolute ethanol solution is marked as a product II;
weighing 1mmol of salicylic acid and 1mmol of triethylamine, dissolving in 15ml of dichloromethane, dissolving 1mmol of acryloyl chloride in 3ml of dichloromethane, placing in a constant pressure dropping funnel, controlling the dropping speed, carrying out ice bath, completing dropping within 1 hour, and stirring for 6 hours; washing twice with 20% dilute hydrochloric acid, washing twice with saturated NaOH solution, washing three times with deionized water, separating liquid, taking an organic phase, drying, and distilling under reduced pressure to obtain a product III;
25mg of product III and 7.5mg of acrylic acid are weighed out and dissolved in 5ml of dichloromethane, stirred and mixed uniformly, 0.5mg of AIBN, N are added2Protecting, reacting at 65 ℃ for 24 hours, adding 50ml of absolute methanol into the reaction solution to generate white precipitate, washing with absolute ethanol twice, and washing with deionized water three times to obtain a product IV;
dissolving the product II in 30ml of DMF, weighing 2mmol of the product IV, dissolving in 10ml of DMF, adjusting the pH to 7 by using 1M of NaOH aqueous solution, stirring for 48 hours in a water bath kettle at 50 ℃, generating a precipitate, standing for 24 hours, carrying out suction filtration, separating out the precipitate, filtering, repeatedly washing by using a small amount of ethanol, and drying to obtain fluorescent nano microspheres, namely the target product;
(2) activation of fluorescent nanospheres
Putting 100 mul of fluorescent nano-microspheres into a 1.5ml EP tube, adding 100 mul of 1mg/ml EDC and 100 mul of 0.6mg/ml NHS, activating for 60min at 37 ℃, centrifuging at high speed of 14000rpm, removing the rest EDC and NHS, and washing with water for three times to obtain a target product;
(3) preparation of fluorescence-labeled urine microalbumin antibody
Incubating 100 μ l of 20 μ g/ml urine microalbumin antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark place to obtain a target product;
(4) preparation of fluorescent-labeled goat anti-chicken IgY antibody
Incubating 100 μ l of 20 μ g/ml goat anti-chicken IgY antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal the residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark to obtain a target product;
(5) preparation of nitrocellulose membranes
Respectively using buffer solutions to respectively draw the urine microalbumin monoclonal antibody and the chicken IgY antibody as a detection line and a quality control line in parallel on a nitrocellulose membrane for coating, wherein the interval between the quality control line and the detection line is 3-5mm, placing the test lines in a drying oven, and drying the test lines at 45 ℃ overnight to obtain a target product;
(6) and sequentially and mutually bonding a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on the PVC base plate in a lap joint manner to obtain a test paper plate, and cutting to obtain the detection test paper strip.
The detection test strip is fixed on the card shell, the surface of the test strip is compressed by a card surface, and the card surface is respectively reserved with a sample adding hole and an observation window at the part corresponding to the sample pad, the combination pad and the nitrocellulose membrane.
The urine microalbumin diluent is phosphate buffer.
The urine microalbumin chip is the same batch of reagent standard curve information and can not be used interchangeably.
Comparative example 2
A urine microalbumin detection kit comprises a urine microalbumin detection card, a urine microalbumin diluent and a urine microalbumin chip:
the urine microalbumin detection card comprises a card shell and a test strip;
the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC base plate, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially stuck on the PVC base plate;
the combination pad contains a urine microalbumin antibody which is marked by fluorescence and a goat anti-chicken IgY antibody which is marked by fluorescence;
the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, the detection line and the quality control line are mutually parallel, the distance is 3-5mm, and the detection line is close to the sample adding hole; the detection line is coated with a urine microalbumin monoclonal antibody, and the quality control line is coated with a chicken IgY antibody.
The preparation method of the europium nano-microsphere fluorescence detection test strip comprises the following steps:
(1) preparation of fluorescent nano-microspheres
Adding 50ml of absolute ethyl alcohol, 3ml of ammonia water and 1ml of water into a three-neck flask in sequence, heating to 40 ℃, keeping the temperature, stirring, quickly adding 1.5ml of tetraethoxysilane, reacting for 7 hours, then adding 1ml of tetraethoxysilane, and continuing to react for 15 hours; centrifuging at high speed, washing with ethanol and water for three times respectively, and redissolving in water to obtain silicon dioxide nanoparticles which are marked as a product I;
weighing 1mmol Eu2O3Placing in a beaker, adding 10ml 20% diluted hydrochloric acid, stirring for dissolving, heating at 80 deg.C for nearly drying, removing excessive HCl, adding 20ml anhydrous ethanol for dissolving to obtain 2mmol EuCl3The absolute ethanol solution is marked as a product II;
4 mmol 1 of benzoylacetone are weighed out and dissolved in 20ml of ethanol, the product II is slowly added dropwise to an ethanol solution of benzoylacetone with stirring at 60 ℃ and the pH is adjusted to 6 with NaOH, a large amount of precipitate appears. Heating, refluxing and stirring for 30min, then dropwise adding 10ml of an ethanol solution containing 2mmol of phenanthroline, continuously stirring for 1h, and drying to obtain a product III;
dissolving the product III in 30ml of DMF, adjusting the pH value to 7 by using 1M NaOH aqueous solution, stirring for 48 hours in a water bath kettle at 50 ℃, standing for 24 hours, performing suction filtration, separating out a precipitate, filtering, repeatedly washing by using a small amount of ethanol, and drying to obtain fluorescent nano microspheres, namely target products;
(2) activation of fluorescent nanospheres
Putting 100 mul of fluorescent nano-microspheres into a 1.5ml EP tube, adding 100 mul of 1mg/ml EDC and 100 mul of 0.6mg/ml NHS, activating for 60min at 37 ℃, centrifuging at high speed of 14000rpm, removing the rest EDC and NHS, and washing with water for three times to obtain a target product;
(3) preparation of fluorescence-labeled urine microalbumin antibody
Incubating 100 μ l of 20 μ g/ml urine microalbumin antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark place to obtain a target product;
(4) preparation of fluorescent-labeled goat anti-chicken IgY antibody
Incubating 100 μ l of 20 μ g/ml goat anti-chicken IgY antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal the residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark to obtain a target product;
(5) preparation of nitrocellulose membranes
Respectively using buffer solutions to respectively draw the urine microalbumin monoclonal antibody and the chicken IgY antibody as a detection line and a quality control line in parallel on a nitrocellulose membrane for coating, wherein the interval between the quality control line and the detection line is 3-5mm, placing the test lines in a drying oven, and drying the test lines at 45 ℃ overnight to obtain a target product;
(6) and sequentially and mutually bonding a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on the PVC base plate in a lap joint manner to obtain a test paper plate, and cutting to obtain the detection test paper strip.
The detection test strip is fixed on the card shell, the surface of the test strip is compressed by a card surface, and the card surface is respectively reserved with a sample adding hole and an observation window at the part corresponding to the sample pad, the combination pad and the nitrocellulose membrane.
The urine microalbumin diluent is phosphate buffer.
The urine microalbumin chip is the same batch of reagent standard curve information and can not be used interchangeably.
The urine microalbumin detection kit prepared in the examples 1-3 and the comparative examples 1-2 is subjected to quantitative detection:
1. precision degree
TABLE 1 results of precision measurements
Figure BDA0003253669640000121
Figure BDA0003253669640000131
2. Accuracy of
TABLE 2 accuracy evaluation test results
Figure BDA0003253669640000132
Figure BDA0003253669640000141
3. Detection limit concentration
TABLE 3 detection limit detection data
Figure BDA0003253669640000142
4. Specificity of
TABLE 4 detection results of specificity test (MALB negative)
Figure BDA0003253669640000143
Figure BDA0003253669640000151
Figure BDA0003253669640000161
TABLE 5 results of the specificity test (MALB concentration 50mg/mL)
Figure BDA0003253669640000162
Figure BDA0003253669640000171
Figure BDA0003253669640000181
5. Linear range
TABLE 6 Linear Range confirmation test data
Figure BDA0003253669640000182
TABLE 7 results of linear range analysis
Figure BDA0003253669640000191
Summary of analytical Performance evaluation
The urine trace protein (MALB) detection kit (quantum dot immunofluorescence chromatography) adopts the examples 1, 2, 3, 1 and 2 to respectively carry out performance evaluation tests of precision, accuracy, minimum detection limit, specificity and linear range on a dry-type fluorescence immunoassay analyzer, and the tests are summarized as follows:
(1) the maximum detection result of the batch precision is 6.57 percent, which meets the requirement that the batch precision is not more than 10 percent; the maximum detection result of precision between the embodiments is 5.62%, which meets the requirement that the precision between the embodiments is not more than 10%.
(2) The maximum relative deviation of the detection of the accuracy reference substance is 3.5 percent, and the maximum relative deviation meets the requirement that the relative deviation is within the range of +/-15 percent;
(3) the concentration value of the detection limit of the MALB zero-concentration calibration product is 3.8mg/L at most, and the requirement that the detection limit is not higher than 5mg/L is met.
(4) The sample with the hemoglobin concentration of 6mg/mL and the bilirubin concentration of 0.5mg/mL does not interfere with the detection result of the reagent. Creatinine, ammonium chloride and urea have no influence on the detection result.
(5) Within the concentration range of 5mg/L-300mg/L, the detection linear correlation coefficient of the kit can meet the requirement that the r is more than or equal to 0.990 for developing a preset target.
The test method comprises the following steps:
1. precision degree
The test method comprises the following steps:
(1) randomly extracting 30 detection cards of the same batch, and respectively detecting enterprise precision reference substances of urine trace protein (MALB). The three horizontal concentration samples were tested 10 times in duplicate and the measured concentrations were recorded.
(2) The three example batches of kits were tested once per batch according to the experimental procedure described above.
Data analysis method
Internal precision: and respectively calculating the average value (M) and the Standard Deviation (SD) of 10 detection results of each concentration sample of each batch of kit, and obtaining the variation coefficient according to the formula CV (SD/M multiplied by 100 percent), wherein the variation coefficient CV (%) meets the requirement of not more than 10 percent.
Batch precision: calculating the average value (M) and the Standard Deviation (SD) of the 30 detection results of the three example batch kits of each concentration sample, and obtaining the coefficient of variation according to the formula CV (SD/M multiplied by 100%), wherein the CV (%) value of the coefficient of variation should meet the requirement that the coefficient of variation is not more than 10%.
2. Accuracy of
The experimental method comprises the following steps:
(1) randomly extracting 9 detection cards of the same batch, respectively detecting enterprise reference substances with urine trace protein (MALB) target concentrations of about 20mg/L, 50mg/L and 200mg/L, and recording the measured concentrations.
(2) Three batches of the kit were tested once per batch according to the experimental procedure described above.
Data analysis method
Calculating the average value (M) of the detection results of 3 times of each concentration sample, and calculating the relative deviation B of the measured concentration according to the formula (1), wherein the result meets the requirement that the relative deviation is within the range of +/-15%.
B＝|(M-T)|/T×100％…………………………………………(1)
In the formula:
b-relative deviation;
m-mean value of measured concentration;
t-calibration concentration.
3. Detection limit
Experimental methods
(1) And respectively using the MALB zero-concentration calibrator as a sample for detection, repeatedly measuring for 20 times, and recording the detection result.
(2) The three example batches of kits were tested once per batch according to the experimental procedure described above.
Data analysis method
And respectively calculating the average value (M) and the Standard Deviation (SD) of the 20 detection results to obtain M +2SD, namely the lowest detection limit.
4. Specificity of
Experimental methods
(1) MALB negative serum added with hemoglobin (concentration of 6mg/mL), bilirubin (concentration of 0.5mg/mL), creatinine (concentration of 0.1mg/mL), and ammonium chloride (concentration of 6mg/mL) urea (concentration of 20mg/mL) was used as an interference sample, and each sample was tested three times; the quality control samples with the target concentration of about 50mg/L MALB added with hemoglobin (the concentration is 6mg/mL), bilirubin (the concentration is 0.5mg/mL), creatinine (the concentration is 0.1mg/mL) and ammonium chloride (the concentration is 6mg/mL) and urea (the concentration is 20mg/mL) are taken as MALB positive interference samples, and each sample is tested for three times.
(2) The three example batches of kits were tested once per batch according to the experimental procedure described above.
Data analysis method
(1) Three MALB negative interference samples are respectively calculated, and the detection result is that the MALB should not be higher than 5 mg/L.
(2) Three MALB positive interference samples are respectively calculated, the average value M of the concentrations calculated three times is calculated, and the deviation of the average value M and 50mg/L is not higher than 15%.
5. Linear range
Experimental methods
Establishment of a Linear Range
The detection is carried out by diluting the MALB antigen to concentration levels of 0mg/L, 2mg/L, 5mg/L, 20mg/L, 50mg/L, 100mg/L, 300mg/L and 600mg/L, respectively using a dry-type fluorescence immunoassay analyzer, the detection is carried out once for each batch of the three batches of the kit, the detection is carried out three times for each concentration point, and the concentration value of each detection is recorded.
Data analysis method
Determination of the Linear Range
And respectively calculating the average value M of the T/C values obtained by three times of detection of each concentration, taking the concentration as an abscissa and the T/C average value M as an ordinate to obtain a linear regression equation y as a + bx, gradually reducing data points according to an experimental result until the requirement that the correlation coefficient r value is more than or equal to 0.990 is met, and taking the concentration range which meets the requirement that the correlation coefficient r value is more than or equal to 0.990 as the linear range of the kit.
Internal reference product and calibration product
1. Sources of reference substances
Since urine micro protein (MALB) has no national reference at present, the reference is prepared internally at present.
Internal reference composition
TABLE 8 constitution of the references
Figure BDA0003253669640000221
2. Sources of calibrators
Since urine micro protein (MALB) has no national reference product at present, the calibrator is prepared internally at present.
Internal calibrator composition
TABLE 9 constitution of calibrator
Figure BDA0003253669640000222
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

Claims (5)

1. The utility model provides a urine microalbumin detect reagent box, includes urine microalbumin detects card, urine microalbumin diluent, urine microalbumin chip, its characterized in that:
the urine microalbumin detection card comprises a card shell and a test strip;
the test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, absorbent paper and a PVC base plate, wherein the sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are sequentially stuck on the PVC base plate;
the combination pad contains a urine microalbumin antibody which is fluorescently labeled and a goat anti-chicken IgY antibody which is fluorescently labeled;
the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, the detection line and the quality control line are mutually parallel, the distance is 3-5mm, and the detection line is close to the sample adding hole; the detection line is coated with a urine microalbumin monoclonal antibody, and the quality control line is coated with a chicken IgY antibody.
2. The kit of claim 1, wherein the test strip is prepared by the following steps:
(1) preparation of fluorescent nano-microspheres
Adding 50ml of absolute ethyl alcohol, 3ml of ammonia water and 1ml of water into a three-neck flask in sequence, heating to 40 ℃, keeping the temperature, stirring, quickly adding 1.5ml of tetraethoxysilane, reacting for 7 hours, then adding 1ml of tetraethoxysilane, and continuing to react for 15-20 hours; centrifuging at high speed, washing with ethanol and water for three times respectively, and redissolving in water to obtain silicon dioxide nanoparticles which are marked as a product I;
weighing 1mmol Eu2O3Placing in a beaker, adding 10ml 20% diluted hydrochloric acid, stirring for dissolving, heating at 80-100 deg.C for drying, removing excessive HCl, adding 20ml anhydrous ethanol for dissolving to obtain 2mmol EuCl3The absolute ethanol solution is marked as a product II;
weighing 4mmo1 benzoylacetone, dissolving in 20ml ethanol, slowly dripping the product II into the ethanol solution of the benzoylacetone under stirring at 60 ℃, adjusting the pH to 6-7 by NaOH, and generating a large amount of precipitate; heating, refluxing and stirring for 30min, then dropwise adding 10ml of an ethanol solution containing 2mmol of phenanthroline, continuously stirring for 1h, and drying to obtain a product III;
weighing 1mmol of salicylic acid and 1mmol of triethylamine, dissolving in 15ml of dichloromethane, dissolving 1mmol of acryloyl chloride in 3ml of dichloromethane, placing in a constant pressure dropping funnel, controlling the dropping speed, carrying out ice bath, completing dropping within 1 hour, and stirring for 6-12 hours; washing twice with 20% dilute hydrochloric acid, washing twice with saturated NaOH solution, washing three times with deionized water, separating liquid, taking an organic phase, drying, and distilling under reduced pressure to obtain a product IV;
weighing 25mg of product IV and 3.5-11mg of acrylic acid, dissolving in 5ml of dichloromethane, stirring, mixing uniformly, adding 0.5mg of AIBN, N2Protecting, reacting at 65 ℃ for 24 hours, adding 50ml of absolute methanol into the reaction solution to generate white precipitate, washing with absolute ethanol twice, and washing with deionized water three times to obtain a product V;
dissolving the product III in 30ml of DMF, weighing 2mmol of the product V, dissolving the product V in 10ml of DMF, adjusting the pH to 6-7 by using 1M of NaOH aqueous solution, stirring the solution in a water bath kettle at the temperature of 50-60 ℃ for 24-48h, allowing the solution to stand for 24h, performing suction filtration, separating out a precipitate, filtering, repeatedly washing the precipitate by using a small amount of ethanol, and drying to obtain fluorescent nano microspheres, namely the target product;
(2) activation of fluorescent nanospheres
Putting 100 mul of fluorescent nano-microspheres into a 1.5ml EP tube, adding 100 mul of 1mg/ml EDC and 100 mul of 0.6mg/ml NHS, activating for 60min at 37 ℃, centrifuging at high speed of 14000rpm, removing the rest EDC and NHS, and washing with water for three times to obtain a target product;
(3) preparation of fluorescence-labeled urine microalbumin antibody
Incubating 100 μ l of 20 μ g/ml urine microalbumin antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark place to obtain a target product;
(4) preparation of fluorescent-labeled goat anti-chicken IgY antibody
Incubating 100 μ l of 20 μ g/ml goat anti-chicken IgY antibody with activated fluorescent nanoparticles at 37 ℃ for 90min, adding 100 μ l of 1% BSA, incubating at 37 ℃ for 60min to seal the residual active groups, centrifuging at 14000rpm at high speed to remove supernatant, and storing in dark to obtain a target product;
(5) preparation of nitrocellulose membranes
Respectively using buffer solutions to respectively draw the urine microalbumin monoclonal antibody and the chicken IgY antibody as a detection line and a quality control line in parallel on a nitrocellulose membrane for coating, wherein the interval between the quality control line and the detection line is 3-5mm, placing the test lines in a drying oven, and drying the test lines at 45 ℃ overnight to obtain a target product;
(6) and sequentially and mutually bonding a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper on the PVC base plate in a lap joint manner to obtain a test paper plate, and cutting to obtain the detection test paper strip.
3. The kit of claim 1 or 2, wherein the test strip is fixed on the card housing, the surface of the test strip is pressed by a card surface, and the card surface is provided with a sample hole and an observation window respectively at the parts corresponding to the sample pad, the combination pad and the nitrocellulose membrane.
4. The kit of claim 1, wherein the urine microalbumin diluent is phosphate buffered saline.
5. The kit of claim 1, wherein the urine microalbumin chip is a same lot of reagent standard curve information and is not interchangeable.
CN202111053687.1A 2021-09-09 2021-09-09 Urine microalbumin detection kit and preparation thereof Pending CN113848326A (en)

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