CN118191344A - 25-Hydroxy vitamin D quantum dot fluorescence detection reagent based on sandwich method and application thereof - Google Patents

25-Hydroxy vitamin D quantum dot fluorescence detection reagent based on sandwich method and application thereof Download PDF

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CN118191344A
CN118191344A CN202410614277.7A CN202410614277A CN118191344A CN 118191344 A CN118191344 A CN 118191344A CN 202410614277 A CN202410614277 A CN 202410614277A CN 118191344 A CN118191344 A CN 118191344A
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sample
antibody
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CN118191344B (en
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何长锋
王学士
谢煜萍
姚丛丛
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Shandong Zifeng Biotechnology Co ltd
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors

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Abstract

The invention belongs to the technical field of analytical chemistry and detection, and particularly relates to a sandwich-method-based 25-hydroxy vitamin D quantum dot fluorescence detection reagent and application thereof. The method effectively overcomes the defects of unsupported whole blood, long analysis time and poor sensitivity of the existing known detection method by optimally combining the detection reagents of 25-OH-VD, has the advantages of high detection sensitivity, low detection limit, wider linear range, simple and convenient operation, high stability, realization of normal-temperature transportation and the like, and has important value for detection of 25-OH-VD and evaluation of drug curative effect.

Description

25-Hydroxy vitamin D quantum dot fluorescence detection reagent based on sandwich method and application thereof
Technical Field
The invention belongs to the technical field of analytical chemistry and detection, and particularly relates to a sandwich-method-based 25-hydroxy vitamin D quantum dot fluorescence detection reagent and application thereof.
Background
Vitamin D is a fat-soluble vitamin essential to the human body. Vitamin D exists in the body mainly in two forms, vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D synthesized by direct sunlight to the skin is transported to the liver by binding to Vitamin D Binding Protein (VDBP), whereas vitamin D obtained from food is absorbed into the blood together with fat in the form of chylomicron through the small intestine wall and then is transported to the liver by binding to lipoprotein or vitamin D binding protein. Vitamin D absorbed in two ways must undergo two hydroxylations in the body to exert a biological effect. First, the enzyme is transported to the liver along with blood, and the hydroxylation of C25 is started in the liver microsome under the action of vitamin D-25-hydroxylase and the participation of NADPH and O 2、Mg2+, so that 25-hydroxy vitamin D (25-OH-VD) with weaker physiological activity is generated. The 25-OH-VD in circulation is then transported to the kidney through blood by combining with a-globulin, and is catalytically converted into the most physiologically active 1, 25-dihydroxyvitamin D (1, 25 (OH) 2 D) (also called calcitriol) in the mitochondria of the kidney through 25-hydroxyvitamin D-1a hydroxylase, and then exerts hormone-like biological effects under the mediation of specific Vitamin D Receptors (VDR) in cells, and participates in various biological processes. 25-OH-VD and 1,25 (OH) 2 D are metabolites that are primarily used to evaluate vitamin D status in vivo, the most prominent metabolite in the circulation being 25-OH-VD and the active metabolite being 1,25 (OH) 2 D.
Vitamin D has wide physiological effects, and the maintenance of normal level 25-hydroxy vitamin D is critical for calcium phosphorus metabolism and bone development, and can not only maintain the stability of calcium and phosphorus concentration, promote bone mineralization, but also induce cell differentiation, inhibit cell proliferation, promote tumor cell apoptosis, and realize immunoregulation. Various related diseases are caused when vitamin D levels are below or above the normal reference range. Under the condition of deficiency of vitamin D, the human body can only absorb about 10% -15% of calcium and 60% of phosphorus, and under the condition of sufficient vitamin D, through binding of 1,25 (OH) 2 D with vitamin receptors, the absorption efficiency of the intestinal tract on calcium and phosphorus can be improved, so that the calcium absorption is increased to 30% -40%, and the phosphorus is increased to about 80%.
Recently, the national academy of medical science recommended that blood levels of 25-OH-VD of greater than 20 ng/mL be beneficial to bone health, and many documents define vitamin D deficiency as serum 25-OH-VD levels below 20 ng/mL, serum 25-OH-VD of less than 21 ng/mL-29 ng/mL, serum 25-OH-VD of more than 30 ng/mL, and serum 25-OH-VD of greater than 150 ng/mL may lead to poisoning. However, since the total amount of 25-OH-VD produced in the body is related to age, race, skin color, living environment, season and living habit, and the individual difference is large, so far, there is no unified standard for the normal reference range of 25-OH-VD in China.
The existing detection methods for 25-OH-VD clinically comprise spectrophotometry, chromatography, chemical reflection method, enzyme-linked immunosorbent assay and the like, and the existing chromatography or liquid mass spectrometry detection methods have the problems of variable vitamin content, long analysis time and poor method specificity during sample processing caused by complex sample processing, and do not support whole blood detection; the existing chemiluminescence method and chromatography mainly rely on the detection principle of the competition method, and have the problems of poor sensitivity and narrow detection range. The limitations of the above detection methods limit the application of the methodology in clinical departments or basic medical facilities.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a sandwich-method-based 25-hydroxy vitamin D quantum dot fluorescence detection reagent and application thereof. Specifically, the detection reagent of the 25-OH-VD is optimally combined, so that the defects of unsupported whole blood, long analysis time and poor sensitivity of the existing known detection method are overcome, and the detection method has the advantages of high detection sensitivity, low detection limit, wider linear range, simplicity and convenience in operation, high stability, capability of realizing normal-temperature transportation and the like, and has important value for detection of the 25-OH-VD and evaluation of the curative effect of the medicine. Based on the above results, the present invention has been completed.
Specifically, the invention relates to the following technical scheme:
The invention provides a sandwich-method-based 25-OH-VD quantum dot fluorescence detection reagent, which at least comprises a diluent for diluting a sample to be detected and a sandwich-method-based 25-OH-VD quantum dot fluorescence detection test strip;
wherein the diluent comprises cyclodextrin, polyethylene glycol, a reducing/denaturing agent, a nonionic surfactant and a denaturing agent.
The cyclodextrin can be any one or more of alpha-cyclodextrin, beta-cyclodextrin or gamma-cyclodextrin and derivatives thereof; further preferably, the cyclodextrin is hydroxypropyl-beta-cyclodextrin.
The molecular weight of the polyethylene glycol is 200-25000, and the polyethylene glycol comprises polyethylene glycol high polymer or polyethylene glycol derivative; further, the polyethylene glycol may be PEG2000, PEG6000 or PEG8000.
The reducing/denaturing agent is one or a combination of more of guanidine hydrochloride, urea, dithiothreitol and mercaptoethanol; further, the denaturant is dithiothreitol.
The nonionic surfactant is selected from one or a combination of a plurality of S9, S17, S19, S20 and S21; further preferably, the nonionic surfactant is S21.
The sample to be measured is a blood sample, specifically, may be a whole blood, serum or plasma sample, and is not specifically limited herein.
The diluent should also maintain a similar osmotic environment to the sample to be tested, thus using physiological saline and/or buffer as solvents; a specific example of the buffer may be disodium hydrogen phosphate-sodium dihydrogen phosphate buffer; preferably, the formula of the diluent is as follows: 0.05-0.15M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 5.5-6.5) contains 0.8-1.0% NaCl, 1-3% hydroxypropyl-beta-cyclodextrin, 1-3% PEG6000, 0.5-0.8% dithiothreitol and 0.4-0.7% S21.
The 25-OH-VD quantum dot fluorescence detection test strip based on the sandwich method comprises a bottom plate, wherein a chromatographic structure is covered on the bottom plate, a sample pad, a bonding pad, a coating film and a water absorption pad are sequentially arranged on the chromatographic structure according to the flowing direction of a sample, the adjacent parts of the components are in overlapping connection, and the adjacent parts of the components are in overlapping connection;
The sample pad is provided with a sample adding hole, and a slow release film is sprayed at the sample adding hole to release sample liquid at fixed time, so that the sample liquid is fully dissociated, and blood cells can be filtered gently.
The binding pad is coated with a mixture of quantum dot marked VDBP and quantum dot marked chicken IgY antibody, the mixture is dissolved and diluted by a compound solution, and then the mixture is sprayed on the binding pad, and the compound solution has the following formula: 0.1 to 0.2M HEPES buffer (pH 7.0 to 7.4) containing 0.8 to 1.0% sodium caseinate, 1 to 2% BSA, 10 to 25% glycerol, 5 to 25% trehalose and 0.1 to 0.3% Proclin300;
In order to ensure the high activity and stability of the quantum dot labeled antibody to the greatest extent, the bonding pad is sprayed and then dried at a low temperature, such as freeze drying.
A detection line (T line) and a quality control line (C line) are sequentially arranged on the coating film along the chromatographic direction; wherein, the T line is coated with an anti-25-OH-VD antibody, and the C line is coated with a goat anti-chicken IgY antibody.
In the test strip, the sample pad, the bonding pad and the coating film are sequentially overlapped, and the water absorption pad and the detection pad are overlapped on the detection pad at the bordering part;
further, the sample pad may be a glass cellulose membrane; the bonding pad may be a polyester cellulose film; the coating film may be a nitrocellulose film; the absorbent pad may be a material having good water absorbing properties, such as a glass fiber material, a cotton pulp material or a mixture of glass fiber and cotton pulp, preferably a cotton pulp material, or a commercially available absorbent material, such as absorbent paper or absorbent board. The present invention is not particularly limited herein.
Furthermore, in the test strip, a bottom plate of the test strip is made of a hydrophobic material with certain hardness; further, the bottom plate is a PVC plate.
The sample pad contains anti-red blood cell antibodies (RBCs) for filtering red blood cells from a whole blood sample; the pretreatment of the sample pad is carried out by spraying RBC pretreatment liquid, and the RBC pretreatment liquid comprises the following formula: 0.05-0.15M HEPES buffer (pH 7-8) containing 2-3% NH 4 Cl, 1-3% sodium caseinate, 2-3% PVP-K30, 160-200 μg/mL anti-erythrocyte antibody (RBC) and 0.3-0.5% Proclin300; spraying the RBC pretreatment liquid with the formula, and drying to obtain the sample pad; in order to retain the activity of RBC, the drying is preferably performed in a low-temperature drying mode, such as a freeze drying mode, and the research discovers that the freeze drying mode after the sample pad is pretreated can not only retain the activity of RBC to the greatest extent, but also seal the vacant sites on the sample pad to avoid adsorbing target substances in the blood sample, thereby achieving the purpose of improving the sensitivity. Meanwhile, considering that most 25-OH-VD exists in a combined state in a sample, small molecules 25-OH-VD are required to be dissociated from the combined protein during detection, in order to accurately control the dissociation time of the sample, and simultaneously, the whole blood sample is not damaged during detection, a slow release film is sprayed at the sample adding hole end of a test strip (the slow release film is sprayed at the corresponding position of the sample adding hole on a sample pad), so that erythrocytes can be caught, and adverse effects on a detection result are reduced. The slow release film is prepared by adding gelatin, hydroxypropyl methylcellulose, D-mannitol and glycerol into water and uniformly stirring; the mass ratio of the gelatin to the hydroxypropyl methylcellulose to the D-mannitol to the glycerol is 1-3:1-1.5:1-1.5: 2-5, wherein the mass volume ratio of the gelatin to the water is 1-3 g:40ml.
The detection reagent takes a blood sample (including whole blood, serum and plasma) as a detection object, and performs quantum dot fluorescence detection on 25-OH-VD in the sample based on a double-antibody sandwich method. The method is significantly different from the conventional chromatography detection method (based on the competition method) in that the method is based on a small molecule double antibody sandwich method, and has higher detection sensitivity and wider detection range; therefore, only a very small amount of sample is needed to finish detection during detection; meanwhile, the detection reagent supports the detection of serum/plasma/whole blood samples, so that the detection reagent is suitable for detecting peripheral blood samples of newborns with severe sample size requirements.
The principle of the method for detecting the 25-hydroxy vitamin D based on the quantum dot fluorescence technology is as follows:
The main reaction part of the quantum dot fluorescence detection test strip for 25-hydroxy vitamin D comprises a sample pad, a binding pad and a coating film: the sample pad is pretreated by RBC, and a slow release film is sprayed at the sample adding hole end; the combination pad is coated with a mixture of quantum dot labeled VD combination protein (VDBP) and quantum dot labeled chicken IgY antibody; and a detection line (T line) and a quality control line (C line) are sequentially arranged on the nitrocellulose membrane (NC membrane) of the coating film, wherein the T line is coated with excessive anti-25-OH-VD antibody, and the C line is coated with excessive goat anti-chicken IgY antibody. In the detection of 25-OH-VD in the kit, a double-antibody sandwich method is adopted, during the detection, firstly, a sample to be detected is mixed with a sample diluent, then the mixture is dripped into the position of a sample adding hole, and the sample mixture stays at the position of the sample adding hole for about 3min for full dissociation due to the action of a slow release film; the mixed solution then enters the sample pad to participate in the chromatography, and the dissociated sample reacts with the labeled antibody on the conjugate pad and moves forward along the nitrocellulose membrane with the sample solution. The quantum dot marked VDBP is combined with 25-OH-VD to be detected in a blood sample, and is captured by a 25-OH-VD complex antibody when passing through a detection line area to obtain a double-antibody sandwich complex, and a T peak is formed; and (3) continuing to move forward the unbound sample solution, capturing the quantum dot labeled chicken IgY by the quality control line antibody goat anti-chicken IgY, forming a C peak, and completing the quality control calibration reaction.
After the display board is finished, the detection card is placed into a detection instrument, the fluorescence value T of the detection line area and the fluorescence value C of the quality control line area are further converted by software to obtain T/C, and the content of 25-OH-VD in the sample to be detected can be obtained through standard curve calculation.
Further, the test strip is prepared by the following method:
1) Preparing a coating film: fixing and scribing a nitrocellulose membrane (NC membrane), respectively taking an anti-25-OH-VD antibody and a goat anti-chicken IgY, respectively coating the positions of a detection line (T) and a quality control line (C) of the NC membrane, and drying.
2) Preparing a bonding pad: and uniformly mixing the quantum dot marked VDBP and the quantum dot marked chicken IgY, spraying the mixture on a glass fiber membrane, and drying to obtain the bonding pad.
The quantum dot marked VDBP and the quantum dot marked chicken IgY antibody are prepared by the following method:
a: adding cadmium selenide quantum dots into MES buffer solution, and activating; then EDC and NHS are added into the mixture, and VDBP or chicken IgY antibody is added into the mixture to carry out light-shielding reaction;
b: b, adding BSA and ethanolamine into the quantum dot marking liquid in the step a, and carrying out light-shielding stirring reaction at a low temperature (such as 4 ℃) to seal;
c: c, centrifuging the marked mixed solution in the step b, and cleaning by using PBS buffer solution as a cleaning solution;
d: c, re-dissolving the marked solution obtained in the step c by using a re-solution, and uniformly mixing to obtain the quantum dot marked VDBP and the quantum dot marked chicken IgY antibody.
Wherein the formulation of the complex solution is as follows: 0.1 to 0.2M HEPES buffer (pH 7.0 to 7.4) containing 0.8 to 1.0% sodium caseinate, 1 to 2% BSA, 10 to 25% glycerol, 5 to 25% trehalose and 0.1 to 0.3% Proclin300;
further, the compound solution comprises the following formula: 0.2M HEPES buffer (pH 7.0), 0.8% sodium caseinate, 1.5% BSA,20% glycerol, 20% trehalose, 0.3% Proclin300.
3) Sample pad pretreatment:
a: preparing RBC pretreatment liquid: 0.05-0.15M HEPES buffer (pH 7-8) containing 2-3% NH 4 Cl, 1-3% sodium caseinate, 2-3% PVP-K30, 160-200 μg/mL anti-erythrocyte antibody (RBC) and 0.3-0.5% Proclin300;
b: c, soaking the sample pad by using the RBC pretreatment liquid prepared in the step a;
c: b, freeze-drying the pretreated sample pad in the step b;
d: preparing a slow release film: the mass ratio of gelatin to hydroxypropyl methyl cellulose to D-mannitol to glycerol is 2:1.5:1.5:3, and the mass volume ratio of gelatin to water is 2g to 40ml;
e: spraying the slow-release membrane liquid prepared in the step d on the sample adding hole position of the sample pad obtained by freeze-drying in the step c, spraying the sample pad at a concentration of 5 mu L/cm, and freeze-drying to obtain the sample pad.
4) And (3) assembling: tearing off the adhesive paper at the corresponding position on the bottom plate, and adhering the water absorbing pad above the NC film of the coating film to partially cover the NC film; bonding the bonding pad under the NC film, and partially covering the bonding pad; and (3) sticking the sample pad below the bonding pad, and partially covering the bonding pad to obtain the assembled large plate.
5) Cutting and packing the shell: and after setting the width of the test strip, cutting to obtain the prepared test strip.
And further, the test strip prepared in the step 5) is positively placed into a card shell, and the card shell is pressed and packaged to obtain the detection card with the shell protection.
In a second aspect of the present invention, there is provided a detection kit comprising the above-described quantum dot fluorescent detection reagent, further, in the detection kit, the outside of the test strip has a housing protection.
In a third aspect of the invention, there is provided a method for detecting the 25-hydroxyvitamin D content in blood based on a sandwich method, which method is based on the quantum dot fluorescent detection reagent according to the first aspect or the detection kit according to the second aspect to obtain the concentration of 25-hydroxyvitamin D in a blood sample of a subject.
Further, the detection method specifically comprises the following steps: and taking a blood sample to be tested, dripping the blood sample into a sample dilution liquid, uniformly mixing, dripping the blood sample into a sample adding hole of a test strip, standing, and detecting the fluorescent content of a detection line and a quality control line in a detection pad after a display board of the test strip is displayed.
Further, the fluorescence content detection mode includes, but is not limited to, fluorescence immunoassay, flow type, fluorescence microscope, confocal microscope, total internal reflection or two-photon microscope, and the like, and the fluorescence detection wavelength is 610nm.
Further, the blood sample panel time was 10min.
In a fourth aspect of the present invention, there is provided the use of a test strip, test kit or test method as described above in any one or more of the following:
1) Detecting the content of 25-hydroxy vitamin D;
2) Preparing a bone metabolism auxiliary diagnosis product;
3) And preparing a product for evaluating the curative effect of the vitamin D medicament.
The product may be a kit, a detection device or a detection apparatus, etc., and is not particularly limited herein.
In addition, the percentages of the raw material components in the diluent, the compound solution and the RBC pretreatment liquid are all mass percentages.
The beneficial technical effects of one or more of the technical schemes are as follows:
1. The 25-hydroxy vitamin D detection reagent provided by the technical scheme is used for detecting small molecules by a double-antibody sandwich method, improves the detection sensitivity and widens the detection range.
25-Hydroxyvitamin D is a small molecular substance, belongs to hapten, has no immunogenicity but has antigenicity, so that the detection is generally carried out in vitro by adopting a competition method, namely the free antigen to be detected in a sample competes for the same limited amount of antibodies together with the antigen immobilized on a solid carrier, and therefore, the detection has poor sensitivity and a narrow detection range.
In order to solve the above-mentioned limitation problem, the invention adopts the recombinant VDBP as the primary antibody, and after quantum dot labeling, the recombinant VDBP is specifically combined with 25-OH-VD to form a "25-OH-VD compound", and adopts the anti-25-OH-VD antibody as the secondary antibody (i.e. detection line antibody), when the sample liquid passes through the detection line area, a double-antibody sandwich structure (T peak) is formed. The sandwich method is used for labeling the antibody in excess and coating the antibody in excess, so that the sandwich method has higher sensitivity and wider detection range, greatly reduces the required amount of a detection sample, and can finish detection by only a trace amount of sample (5 mu L).
2. The slow release film is arranged at the sample adding hole of the test strip: the slow release film can form effective physical barrier in a short time, so that after the sample is mixed with the diluent, the sample is reacted for 3min at the position of the sample adding hole, and the 25-OH-VD in the combined state in the sample is dissociated from the combined protein.
3. After the sample pad is pretreated by the RBC-containing buffer solution, the freeze drying mode is adopted, the activity of RBC is reserved to the greatest extent, the vacant sites on the blood filtering membrane can be closed, and the adsorption of target objects (25-OH-VD) in the blood sample is avoided, so that the purpose of effectively filtering red blood cells is achieved, the detection of the whole blood sample is realized, and the detection results of the whole blood sample, serum and plasma sample have good consistency.
4. The characteristic of the small molecule is unstable, and the kit is carried out in a freeze-drying mode on the membrane drawing and the spray pad involved in the antibody immobilization process, so that the stability of the antigen and the antibody is ensured to the greatest extent.
Meanwhile, the test paper provided by the technical scheme has good stability, and can be transported and stored at normal temperature, so that the test paper has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of a test strip according to the present invention; wherein, 1-sample pad, 2-combination pad, 3-NC membrane, 4-absorbent pad, 5-bottom plate, 6-slow release membrane, 7-detection line, 8-quality control line.
FIG. 2 is a schematic view of the outer housing of the test strip of the present invention; wherein, 9-sample adding hole and 10-observation window (window).
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of biology within the skill of the art, and are fully explained in the literature.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
The embodiment provides a sandwich-method-based quantum dot fluorescence detection method for 25-hydroxy vitamin D, which comprises the steps that the structure of a detection test strip is shown in a figure 1, the test strip comprises a bottom plate 5 and a chromatographic structure, and the bottom plate 5 bears the chromatographic structure; the bottom plate 5 is a PVC plate.
The chromatographic structure is provided with a sample pad 1, a combination pad 2, an NC membrane 3 and a water absorption pad 4 from a sample adding end to the sample chromatographic direction, the sample pad 1, the combination pad 2 and the NC membrane 3 are sequentially overlapped, and the water absorption pad 4 is overlapped on the NC membrane 3 at a part contacting the NC membrane 3.
In this embodiment, the method for detecting the fluorescence of the quantum dot by using the 25-hydroxyvitamin D of the detection test strip comprises the following specific operation steps:
1) Preparing a coating film:
coating: fixing the non-sample-application surface of the NC film at a set position of a film-scribing and metal spraying instrument, taking an anti-25-OH-VD antibody with the concentration of 1.8mg/mL and a sheep anti-chicken IgY antibody with the concentration of 0.9mg/mL, scribing by using a three-dimensional film-scribing and metal spraying instrument, respectively coating the anti-25-OH-VD antibody and the sheep anti-chicken IgY antibody at the positions of a detection line T and a quality control line C of the NC film, and freeze-drying for 48 hours.
2) Preparation of bond pads
(A) Quantum dot-marked VDBP
A: 100 mu L of cadmium selenide quantum dot is taken and added into 4mL of MES buffer solution (pH 6.0) with the ionic strength of 50mM, 20mg/mL of EDC and 20mg/mL of NHS are added into the solution, 100 mu L of each of 20mg/mL of EDC and 5mg/mL of VDBP are added into the solution, the solution is reacted for 3 hours at room temperature in a dark place;
b: adding BSA with the final concentration of 6mg/mL and ethanolamine with the final concentration of 1mg/mL into the quantum dot marking liquid prepared in the step a, and carrying out light-shielding stirring reaction for 4 hours at 4 ℃ for sealing;
c: transferring the marked mixed solution in the step b into a centrifuge tube, centrifuging at 14000g/min for 15min, and discarding the supernatant; adding 4mL of 50mM PBS buffer solution (pH 7.4) serving as a cleaning solution into the centrifuge tube, blowing uniformly, centrifuging for 15min again at 14000g/min, and discarding the supernatant to obtain the labeled antibody;
d: c, re-dissolving the labeled antibody obtained in the step c by using a re-solution, carrying out ultrasonic treatment and uniformly mixing to obtain the purified quantum dot labeled folic acid antibody.
(B) Quantum dot marked chicken IgY
A: taking 100 mu L of cadmium selenide quantum dots, adding the cadmium selenide quantum dots into 4mL of MES buffer solution (pH 6.0) with the ionic strength of 50mM, adding 100 mu L of 20mg/mL EDC and 20mg/mL NHS into the MES buffer solution, respectively adding 500 mu L of 5mg/mL chicken IgY antibody into the EDC and carrying out light-shielding reaction for 3 hours at room temperature;
b: adding casein with the final concentration of 6mg/mL and ethanolamine with the final concentration of 1mg/mL into the quantum dot marking liquid prepared in the step a, and carrying out light-shielding stirring reaction for 4 hours at 4 ℃ for sealing;
c: transferring the marked mixed solution in the step b into a centrifuge tube, centrifuging at 12000g/min for 15min, and discarding the supernatant; adding 4mL of 50mM PBS buffer solution (pH 7.4) serving as a cleaning solution into the centrifuge tube, blowing uniformly, centrifuging at 12000g/min for 15min again, and discarding the supernatant to obtain the labeled antibody;
d: c, re-dissolving the marked antibody obtained in the step c by using a re-solution, carrying out ultrasonic treatment and uniformly mixing to obtain the purified quantum dot marked chicken IgY antibody.
The formula of the complex solution is as follows: 0.2M HEPES buffer (pH 7.0), 0.8% sodium caseinate, 1.5% BSA,20% glycerol, 20% trehalose, 0.3% Proclin300.
(C) Spray pad
The re-dissolved quantum dot marked VDBP and quantum dot marked chicken IgY are mixed according to the following ratio of 1:1, spraying the mixture on a glass fiber mat according to the volume of 3 mu L/cm by using a three-dimensional scribing metal spraying instrument, and freeze-drying the mixture for 48 hours to obtain the bonding mat.
3) Sample pad pretreatment
A: preparing RBC pretreatment liquid: 0.1M HEPES buffer (pH 7.5) containing 2.5% NH 4 Cl, 2% sodium caseinate, 2.5% PVP-K30 and 180. Mu.g/mL anti-erythrocyte antibody (RBC) and 0.4% Proclin300;
b: c, soaking the blood filtering membrane by using the RBC pretreatment liquid prepared in the step a for 1h at room temperature;
c: c, drying the pretreated blood filtering membrane in the step b by using a freeze dryer, and freeze-drying for 48 hours to obtain the pretreated blood filtering membrane;
d: preparing a slow-release membrane solution: the mass ratio of gelatin, hydroxypropyl methylcellulose, D-mannitol, glycerol and water is 2:1.5:1.5:3:40, and the preparation is ready to use; the use of the slow release film is beneficial to improving the stability of a sample to be detected and the compatibility of a detection system, thereby further improving the detection sensitivity and the like.
E: spraying the slow-release membrane liquid prepared in the step d on the sample adding hole position of the sample pad obtained by freeze-drying in the step c, spraying the sample pad at a concentration of 5 mu L/cm, and freeze-drying to obtain the sample pad after the treatment.
4) And (3) assembling:
tearing off the adhesive paper at the corresponding position on the bottom plate, adhering the water absorbing paper at the corresponding position above the NC film, and covering the NC film by 1mm; bonding the bonding pad under the NC film, and covering the bonding pad by 1mm; and (3) sticking the sample pad below the bonding pad, and covering the bonding pad by 2mm to obtain the assembled large plate.
5) Cutting and packing the shell: and (3) after the width of the test strip is set by using a microcomputer automatic chopper, cutting to obtain the prepared test strip, placing the test strip in a card shell in the forward direction, pressing the shell for packaging to obtain a detection card with shell protection (shown in figure 2), and placing the detection card in an aluminum foil bag for sealing to obtain the detection card of the single 25-hydroxy vitamin D.
6) Preparing a sample diluent. According to the optimal formulation, 0.1M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 5.8) contained 0.9% NaCl,2% hydroxypropyl-beta-cyclodextrin, 2% PEG6000,0.8% dithiothreitol and 0.5% S21.
7) For use in detecting 25-hydroxyvitamin D in a sample, the detection method comprises the steps of:
a. mu.L of the sample to be tested (serum/plasma/whole blood) is sucked up and added into 100 mu.L of the sample diluent to be mixed uniformly for standby.
B. and adding 50 mu L of diluted sample into the sample adding hole of the detection card, inserting the sample into a fluorescence immunoassay analyzer after the display board is subjected to 10min, detecting the fluorescence signal intensity of the detection card, respectively representing the fluorescence values of the detection line and the quality control line by T, C, providing the finally obtained detection result in a ratio form (namely T/C is taken as an output result), substituting the ratio of the detection result into a standard curve, and fitting the concentration of 25-hydroxy vitamin D in the sample to be detected.
Comparative example 1
The preparation method is the same as in example 1, except that no denaturing agent is added when the sample dilution of the 25-hydroxyvitamin D assay kit is prepared.
Comparative example 2
The preparation method is the same as in example 1, except that cyclodextrin substances are not added when the sample diluent of the 25-hydroxy vitamin D detection kit is prepared.
Comparative example 3
The preparation method is the same as in example 1, except that the drying of the coating film, the binding pad and the sample of the 25-hydroxyvitamin D detection kit adopts a more conventional drying mode at 37 ℃ instead of a freeze-drying mode.
Comparative example 4
The preparation method is the same as in example 1, except that the slow-release membrane liquid is not sprayed at the position of the sample adding hole of the sample pad.
Effect verification
1. Example 1 detection of clinical samples
As shown in Table 1, the coincidence rate of the detection 25-OH-VD result in example 1 and the clinical detection 25-OH-VD result can reach 100%, and the correlation is as follows: 0.992.
Table 1 comparison of the test results of example 1 with clinical results
2. Comparative example 1 detection of clinical samples
As shown in Table 2, the coincidence rate of the detected 25-OH-VD result and the clinically detected 25-OH-VD result in comparative example 1 can reach 68%, and the correlation is 0.852.
Table 2 comparison of test results with clinical results for comparative example 1
3. Comparative example 2 detection of clinical samples
As shown in Table 3, the correlation between the detection result of 25-OH-VD in comparative example 2 and the negative-positive coincidence rate of 25-OH-VD in clinical detection is 56%, which is 0.31; and the whole blood sample tested in comparative example 2 showed severe hemolysis.
TABLE 3 comparison of test results with clinical results for comparative example 2
4. Comparative example 3 detection of clinical samples
As shown in Table 4, the coincidence rate of the detected 25-OH-VD result and the clinically detected 25-OH-VD result in comparative example 3 can reach 84%, and the correlation can reach 0.962.
Table 4 comparison of test results with clinical results for comparative example 3
5. Comparative example 4 detection of clinical samples
As shown in Table 5, the coincidence rate of the detected 25-OH-VD result and the clinically detected 25-OH-VD result in comparative example 4 can reach 68%, and the correlation can reach 0.918.
Table 5 comparison of test results with clinical results for comparative example 4
6. The technical index of the test strip in the invention is as follows:
accuracy: the relative deviation is not more than +/-5%;
repeatability: CV is not more than + -5%;
Detection limit: not higher than 1.5ng/mL;
linear range: the correlation r is more than or equal to 0.990 in the range of [2, 200] ng/mL.
7. Test strip stability verification
3 Different batches of test strips prepared as in example 1 were tested with samples of known 25-OH-VD concentration of 27.8ng/mL, and the results are shown in Table 6.
Table 6 test strip stability test results
The method has the advantages of good normal-temperature storage stability, simple and convenient operation and realization of normal-temperature transportation.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (7)

1. The quantum dot fluorescence detection reagent for the 25-hydroxy vitamin D based on the sandwich method is characterized by at least comprising a diluent for diluting a sample to be detected and a quantum dot fluorescence detection test strip based on the 25-OH-VD of the sandwich method;
The formula of the diluent is as follows: 0.05-0.15M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution contains 0.8-1.0% NaCl, 1-3% hydroxypropyl-beta-cyclodextrin, 1-3% PEG6000, 0.5-0.8% dithiothreitol and 0.4-0.7% S21;
The quantum dot fluorescence detection test strip for the 25-hydroxy vitamin D comprises a bottom plate, wherein a chromatographic structure is covered on the bottom plate, and the chromatographic structure is sequentially provided with a sample pad, a bonding pad, a coating film and a water absorption pad according to the flowing direction of a sample, and the adjacent parts of the components are in overlapping connection;
The binding pad is coated with a mixture of quantum dot marked VDBP and quantum dot marked chicken IgY antibody, the mixture is dissolved and diluted by a compound solution, and then the mixture is sprayed on the binding pad, and the compound solution has the following formula: 0.1-0.2M HEPES buffer solution contains 0.8-1.0% of casein sodium, 1-2% of BSA, 10-25% of glycerol, 5-25% of trehalose and 0.1-0.3% of Proclin300;
a detection line T line and a quality control line C line are sequentially arranged on the coating film along the chromatographic direction; wherein, the T line is coated with an anti-25-OH-VD antibody, and the C line is coated with a goat anti-chicken IgY antibody.
2. The quantum dot fluorescent test reagent of claim 1, wherein the sample to be tested is a blood sample, including a whole blood, serum or plasma sample.
3. The quantum dot fluorescent detection reagent according to claim 1, wherein the sample pad, the binding pad and the coating film are sequentially overlapped, and the water absorbing paper and the detection pad are overlapped on the detection pad at the abutting part;
the sample pad is a glass cellulose membrane; the bonding pad is a polyester cellulose film; the coating film is a nitrocellulose film; the water absorption pad is made of glass fiber materials, cotton pulp materials, mixed materials of glass fibers and cotton pulp, water absorption paper or a water absorption plate;
the bottom plate is a PVC plate;
The sample pad is prepared by spraying RBC pretreatment liquid, and the RBC pretreatment liquid comprises the following formula: the HEPES buffer solution with the concentration of 0.05-0.15M contains 2-3% of NH 4 Cl, 1-3% of sodium caseinate, 2-3% of PVP-K30, 160-200 mug/mL of anti-erythrocyte antibody and 0.3-0.5% of Proclin300.
4. The quantum dot fluorescence detection reagent according to any one of claims 1 to 3, wherein the test strip is prepared by the following method:
1) Preparing a coating film: fixing and scribing the nitrocellulose membrane, respectively taking an anti-25-OH-VD antibody and a goat anti-chicken IgY, respectively coating the positions of a detection line T and a quality control line C of the nitrocellulose membrane, and freeze-drying;
2) Preparing a bonding pad: uniformly mixing the quantum dot marked VDBP and the quantum dot marked chicken IgY, spraying the mixture on a glass fiber pad, and drying to obtain the bonding pad;
3) Sample pad pretreatment:
4) And (3) assembling:
Tearing off the adhesive paper at the corresponding position on the bottom plate, and adhering the water absorbing pad above the nitrocellulose membrane of the coating film to partially cover the nitrocellulose membrane; bonding the bonding pad under the nitrocellulose membrane, and partially covering the bonding pad; sticking the blood filtering film below the bonding pad, and partially covering the bonding pad to obtain an assembled large plate;
5) Cutting and packing the shell: and after setting the width of the test strip, cutting to obtain the prepared test strip.
5. The quantum dot fluorescence detection reagent according to claim 4, wherein the quantum dot labeled VDBP or quantum dot labeled chicken IgY antibody is prepared by the following method:
a: adding cadmium selenide quantum dots into MES buffer solution, and activating; then EDC and NHS are added into the mixture, and VDBP or chicken IgY antibody is added into the mixture to carry out light-shielding reaction;
b: c, adding casein and ethanolamine into the quantum dot marking liquid in the step a, and carrying out light-shielding stirring reaction at 4 ℃ for sealing;
c: c, centrifuging the marked mixed solution in the step b, and cleaning by using PBS buffer solution as a cleaning solution;
d: c, re-dissolving the marked solution obtained in the step c by using a re-solution, and uniformly mixing to obtain a quantum dot marked VDBP or a quantum dot marked chicken IgY antibody;
Wherein, the compound solution comprises the following components: 0.1 to 0.2M HEPES buffer solution contains 0.8 to 1.0% of casein sodium, 1 to 2% of BSA, 10 to 25% of glycerol, 5 to 25% of trehalose and 0.1 to 0.3% of Proclin300.
6. A detection kit comprising the quantum dot fluorescent detection reagent of any one of claims 1-5.
7. Use of the quantum dot fluorescent detection reagent of any one of claims 1-5 or the detection kit of claim 6 in any one or more of the following:
1) Preparing a bone metabolism auxiliary diagnosis product;
2) And preparing a product for evaluating the curative effect of the vitamin D medicament.
CN202410614277.7A 2024-05-17 2024-05-17 25-Hydroxy vitamin D quantum dot fluorescence detection reagent based on sandwich method and application thereof Active CN118191344B (en)

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