CN113533282A - Biotin quantitative determination method based on homogeneous phase time-resolved fluorescence - Google Patents
Biotin quantitative determination method based on homogeneous phase time-resolved fluorescence Download PDFInfo
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims abstract description 284
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 8
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- 108010067770 Endopeptidase K Proteins 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 239000007836 KH2PO4 Substances 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 239000002736 nonionic surfactant Substances 0.000 description 4
- 239000011698 potassium fluoride Substances 0.000 description 4
- 235000003270 potassium fluoride Nutrition 0.000 description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 3
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a biotin quantitative determination method based on homogeneous phase time-resolved fluorescence, and belongs to the technical field of in vitro diagnosis homogeneous phase time-resolved fluorescence. The invention provides a method for quantitatively determining biotin in a sample based on homogeneous phase time-resolved fluorescence, which comprises the enzyme digestion step, wherein the enzyme digestion step comprises the following steps: mixing the sample with protease, and performing enzymolysis to obtain an enzyme-digested sample; the method is based on homogeneous phase time-resolved fluorescence, and the homogeneous phase time-resolved fluorescence can be monitored by time resolution, so that a fluorescence signal can be easily distinguished from a short-life fluorescence background, and further background light interference is effectively avoided, and a quantitative determination result is more accurate; the method effectively avoids the difference of the biotinylation level of the protein and the deviation of the steric hindrance to the detection result of the immobilized biotin through enzyme digestion, and further improves the accuracy of the quantitative detection result; the detection limit of the method for quantitatively determining the biotin is only 0.03nM, and the determination sensitivity is extremely high.
Description
Technical Field
The invention relates to a biotin quantitative determination method based on homogeneous phase time-resolved fluorescence, and belongs to the technical field of in vitro diagnosis homogeneous phase time-resolved fluorescence.
Background
Biotin is a small molecule in the B-group of vitamins, is present in every living cell and plays an important biochemical role. Biotin can be coupled to a variety of agents, such as proteins, polypeptides, DNA, ligands, quantum dots, and nanoparticlesParticles, and the like. The biological activity of these agents is hardly affected by biotin conjugation. Moreover, biotin has highly selective and stable interactions with avidin and its homologs. The interaction between biotin and avidin is the strongest non-covalent interaction known at present, and the affinity constant is as high as 1015mol/L. In addition to the commonly used streptavidin, more and more new streptavidin variants are being designed to meet different experimental properties and requirements in vitro and in vivo studies. Meanwhile, various chemical and enzymatic biotinylation techniques based on the biotin-streptavidin system can be applied to various experiments, such as signal amplification and target separation. In addition, biotin is widely applied to the in vitro diagnosis industry as a marker of antibodies, nucleic acids, nanoparticles and the like, so that the quantitative determination of the concentration of biotin has important potential value.
The present inventors have succeeded in studying ELISA (see in particular: Yuo-Driving Chang, C. -H.W., Re-Jiin Chang, David Shiutan, Determination of biochemical considerations by a comprehensive enzyme-linked immunological assay (ELISA) method, J.Biocher. Biophys. methods 1994,29, 321. 329.), Chemiluminescence (see in particular: Xing-guiding, T.Z.S.A.S., Determination of biological in Pharmaceutical Formulations by a laboratory catalyst system, luminescence-CdTe Nanoparticles Chemistry system, Chemistry, research, 28 (see in particular: J.Biotech., J.Biophys.Biophys.S., 11, J.Biophyr. Biophyr. Biophys.12, J.Biophy.Biophy.Biophy.S.; Determination of biochemical assays by Biophys.P.P.P.S.; J.Biophys.S., T.S.S.S., T.S., C.S.S.12, 28, C.C.S.J.J., the limit of detection (LOD) and detection range measured can range from nanomolar to micromolar.
However, with the development of in vitro diagnostic techniques, the sensitivity and accuracy of biotin quantitative determination in many special scenes are increasingly required, and the immunoassay-based biotin quantitative determination methods such as ELISA, chemiluminescence and electrochemiluminescence techniques have not been able to meet the high requirements of these special scenes on the sensitivity and accuracy of biotin quantitative determination. For example, immunoassay techniques using biotin as a labeling molecule for antibodies, nucleic acids, quantum dots, nanoparticles, and the like, currently occupy a large portion of the market in the in vitro diagnostic industry. If the biotinylation level of the reagent can be accurately quantified in the initial development stage of the biotin-labeled diagnostic reagent, the development direction of the reagent can be effectively guided, and the development period is greatly shortened. Also, in a biotin-streptavidin based immunoassay, biotin present in an unknown sample can compete with biotin-labeled reagents, thereby interfering with the test results. For another example, biotin has been added to some over-the-counter medicines in the last few years and is used by an increasing number of people for cosmetic needs, such as hair, nail and skin care. Frequent exposure or consumption of biotin treatments may indirectly or directly result in elevated concentrations of biotin in the blood, leading to misdiagnosis of clinical blood analyses. Moreover, biotin itself is an indispensable vitamin involved in human metabolism, and the concentration of biotin in normal human plasma is not more than 2.05 nM. Accurate monitoring of the level of biotin in plasma can effectively screen the genetic metabolic diseases of biotin deficiency and guide patients to take biotin reasonably. The above-described special scenarios all require highly sensitive and highly accurate detection of samples with lower biotin levels to determine biotin interference based on immunoassay of biotin-streptavidin and to guide in vitro diagnostic reagent development and clinical biotin treatment.
Furthermore, immunoassay-based biotin quantitative determination methods such as ELISA, chemiluminescence, and electrochemiluminescence techniques have some drawbacks. For example, ELISA requires enzyme substrates and additional washing, with assay cycles up to several hours. For example, quantitative biotin measurement methods based on immunoassays such as ELISA, chemiluminescence, and electrochemiluminescence are based on detection of an instantaneous signal, and are susceptible to interference from spontaneous luminescence of reagents, substrates, and solvents, which causes variation in results.
Therefore, it is highly desirable to find a method for quantitative determination of biotin, which has high sensitivity, good accuracy, simple procedure and short measurement period.
Disclosure of Invention
In order to solve the problems, the invention provides a method for quantitatively determining biotin in a sample based on homogeneous phase time-resolved fluorescence, which comprises an enzyme digestion step; the enzyme digestion step is as follows:
and mixing the sample with protease, and carrying out enzymolysis to obtain the enzyme-digested sample.
In one embodiment of the invention, the protease is a serine protease.
In one embodiment of the invention, the serine protease is at least one of proteinase K, chymotrypsin, trypsin and elastase.
In one embodiment of the invention, the protease is a protease solution with a concentration of 50-100. mu.g/mL.
In one embodiment of the invention, the solvent of the protease solution is water, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), Tris (hydroxymethyl) aminomethane (Tris), NaH2PO4&Na2HPO4And K2HPO4&KH2PO4At least one of (1).
In one embodiment of the present invention, the enzymatic hydrolysis is: carrying out enzymolysis for 15-120 min at 55-65 ℃.
In one embodiment of the present invention, the enzymatic hydrolysis is: enzymolysis is carried out for 2h at 700rpm and 58 ℃.
In one embodiment of the invention, the mixing volume ratio of the sample to the protease is 100-200: 1.
In one embodiment of the invention, the mixing volume ratio of the sample to the protease is 150: 1.
In one embodiment of the present invention, the enzyme digestion step further comprises an incubation step; the incubation steps are as follows:
firstly, mixing the enzyme-digested sample with a working reagent 1 marked by a fluorescent group 1, and then incubating to obtain an incubation liquid 1, and then mixing the incubation liquid 1 with a working reagent 2 marked by a fluorescent group 2, and then incubating to obtain an incubation liquid 2.
In one embodiment of the invention, the use isThe working reagent 1 marked by the fluorescent group 1 is allophycocyanin-marked biotin and Eu3+At least one of labeled streptavidin.
In one embodiment of the present invention, when the working reagent 1 labeled with the fluorophore 1 is allophycocyanin-labeled biotin, the working reagent 2 labeled with the fluorophore 2 is Eu3+Labeled streptavidin;
when the working reagent 1 labeled with the fluorescent group 1 is Eu3+When streptavidin is marked, the working reagent 2 marked by the fluorescent group 2 is biotin marked by allophycocyanin.
In one embodiment of the present invention, the allophycocyanin-labeled biotin is an allophycocyanin-labeled biotin solution with a biotin concentration of 1-20 nM.
In one embodiment of the present invention, the allophycocyanin-labeled biotin is an allophycocyanin-labeled biotin solution with a biotin concentration of 12.5 nM.
In one embodiment of the present invention, the Eu3+The marked streptavidin is Eu with the streptavidin concentration of 0.2-4 nM3+Labeled streptavidin solution.
In one embodiment of the present invention, the Eu3+The labeled streptavidin is Eu with the streptavidin concentration of 2.5nM3+Labeled streptavidin solution.
In one embodiment of the invention, the allophycocyanin-labeled biotin solution and Eu3+The solvent of the marked streptavidin solution is working solution; the components of the working solution comprise a buffering agent, a salt and a protein protective agent.
In one embodiment of the invention, the buffer is 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), Tris (hydroxymethyl) aminomethane (Tris), NaH2PO4&Na2HPO4And K2HPO4&KH2PO4At least one of; the salt is potassium fluoride; the protein protective agent is bovine serum albumin.
In one embodiment of the invention, the working solution comprises 2-60 g/L of buffer, 5-100 g/L of salt and 0.4-10 g/L of protein protective agent.
In one embodiment of the invention, the working solution comprises 5-15 g/L of buffer, 10-30 g/L of salt and 1-3 g/L of protein protective agent.
In one embodiment of the invention, the components of the working fluid comprise 11.916g/L buffer, 23.24g/L salt, and 2g/L protein protectant.
In one embodiment of the present invention, the components of the working fluid further comprise a surfactant.
In one embodiment of the present invention, the working fluid further comprises 0.1 to 1ml/L of a surfactant.
In one embodiment of the present invention, the working fluid further comprises 0.5ml/L of a surfactant.
In one embodiment of the invention, the surfactant is a nonionic surfactant.
In one embodiment of the invention, the non-ionic surfactant is at least one of tween 20, tween 40, tween 60, tween 80, triton 100 and triton 114.
In one embodiment of the present invention, the components of the working fluid further comprise a preservative.
In one embodiment of the present invention, the working solution further comprises 0.1 to 1ml/L of a preservative.
In one embodiment of the invention, the components of the working solution further comprise 0.5ml/L preservative.
In one embodiment of the invention, the preservative is a proclin series biological preservative.
In one embodiment of the invention, the proclin-series biological preservative is at least one of proclin150, proclin200, proclin300 and proclin 5000.
In one embodiment of the present invention, the pH of the working fluid is 6.0 to 8.0.
In one embodiment of the invention, the pH of the working fluid is 7.3.
In one embodiment of the present invention, the pH of the working fluid is adjusted using an aqueous sodium hydroxide solution having a mass concentration of 8%.
In one embodiment of the invention, the incubation is: incubating for 5-30 min at 25-43 ℃.
In one embodiment of the invention, the incubation is: incubate at 37 ℃ for 10 min.
In one embodiment of the invention, the mixing volume ratio of the enzyme-digested sample to the working reagent 1 labeled with the fluorescent group 1 is 2.5-10: 5-9.
In one embodiment of the invention, the mixing volume ratio of the enzyme-digested sample to the working reagent 1 labeled with the fluorophore 1 is 2: 1.
In one embodiment of the invention, the mixing volume ratio of the incubation liquid 1 and the working reagent 2 marked by the fluorescent group 2 is 11.5-15: 5-9.
In one embodiment of the present invention, the mixing volume ratio of the incubation liquid 1 and the working reagent 2 labeled with the fluorophore 2 is 3: 1.
In one embodiment of the present invention, the incubation step is followed by an assay step; the determination steps are as follows:
and (3) carrying out fluorescence excitation on the incubation liquid 2, then determining the fluorescence signal of the incubation liquid 2, and finally calculating to obtain the concentration of the biotin in the sample according to the fluorescence signal and the relation between the fluorescence signal and the concentration of the biotin.
In one embodiment of the invention, the excitation wavelength of the fluorescence signal is 320 nm.
In one embodiment of the invention, the emission wavelengths of the fluorescent signal are 620nm and 665 nm.
In one embodiment of the invention, the calculation is: bringing the fluorescence signal into a fitting equation obtained by fitting according to the relation between the fluorescence signal and the biotin concentration; the fitting equation is as follows:
Y＝36311.3/[1+(x/5.398)^-1.395]+975.7；
in the fitting equation, x is the concentration of biotin in the sample, unit nM, and Y is the signal value S; the calculation formula of the signal value S is as follows:
S＝(λ665nm/λ620nm)^10000；
in the formula, λ665nmThe fluorescence signal of the incubation liquid 2 under the emission wavelength of 665nm is AU; lambda [ alpha ]620nmThe fluorescence signal of incubation fluid 2 at an emission wavelength of 620nm is given in AU.
The invention also provides a kit for quantitatively determining biotin in a sample, which comprises protease, a working reagent 1 marked by the fluorescent group 1 and a working reagent 2 marked by the fluorescent group 2.
In one embodiment of the invention, the protease is a serine protease.
In one embodiment of the invention, the serine protease is at least one of proteinase K, chymotrypsin, trypsin and elastase.
In one embodiment of the invention, the protease is a protease solution with a concentration of 50-100. mu.g/mL.
In one embodiment of the invention, the solvent of the protease solution is water, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), Tris (hydroxymethyl) aminomethane (Tris), NaH2PO4&Na2HPO4And K2HPO4&KH2PO4At least one of (1).
In one embodiment of the present invention, the working reagent 1 labeled with the fluorescent group 1 is allophycocyanin-labeled biotin and Eu3+At least one of labeled streptavidin.
In one embodiment of the present invention, when the working reagent 1 labeled with the fluorophore 1 is allophycocyanin-labeled biotin, the working reagent 2 labeled with the fluorophore 2 is Eu3+Labeled streptavidin;
when the working reagent 1 labeled with the fluorescent group 1 is Eu3+When labeled streptavidin is used, theThe working reagent 2 marked by the fluorescent group 2 is biotin marked by allophycocyanin.
In one embodiment of the present invention, the allophycocyanin-labeled biotin is an allophycocyanin-labeled biotin solution with a biotin concentration of 1-20 nM.
In one embodiment of the present invention, the allophycocyanin-labeled biotin is an allophycocyanin-labeled biotin solution with a biotin concentration of 12.5 nM.
In one embodiment of the present invention, the Eu3+The marked streptavidin is Eu with the streptavidin concentration of 0.2-4 nM3+Labeled streptavidin solution.
In one embodiment of the present invention, the Eu3+The labeled streptavidin is Eu with the streptavidin concentration of 2.5nM3+Labeled streptavidin solution.
In one embodiment of the invention, the allophycocyanin-labeled biotin solution and Eu3+The solvent of the marked streptavidin solution is working solution; the components of the working solution comprise a buffering agent, a salt and a protein protective agent.
In one embodiment of the invention, the buffer is 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), Tris (hydroxymethyl) aminomethane (Tris), NaH2PO4&Na2HPO4And K2HPO4&KH2PO4At least one of; the salt is potassium fluoride; the protein protective agent is bovine serum albumin.
In one embodiment of the invention, the working solution comprises 2-60 g/L of buffer, 5-100 g/L of salt and 0.4-10 g/L of protein protective agent.
In one embodiment of the invention, the working solution comprises 5-15 g/L of buffer, 10-30 g/L of salt and 1-3 g/L of protein protective agent.
In one embodiment of the invention, the components of the working fluid comprise 11.916g/L buffer, 23.24g/L salt, and 2g/L protein protectant.
In one embodiment of the present invention, the components of the working fluid further comprise a surfactant.
In one embodiment of the present invention, the working fluid further comprises 0.1 to 1ml/L of a surfactant.
In one embodiment of the present invention, the working fluid further comprises 0.5ml/L of a surfactant.
In one embodiment of the invention, the surfactant is a nonionic surfactant.
In one embodiment of the invention, the non-ionic surfactant is at least one of tween 20, tween 40, tween 60, tween 80, triton 100 and triton 114.
In one embodiment of the present invention, the components of the working fluid further comprise a preservative.
In one embodiment of the present invention, the working solution further comprises 0.1 to 1ml/L of a preservative.
In one embodiment of the invention, the components of the working solution further comprise 0.5ml/L preservative.
In one embodiment of the invention, the preservative is a proclin series biological preservative.
In one embodiment of the invention, the proclin-series biological preservative is at least one of proclin150, proclin200, proclin300 and proclin 5000.
In one embodiment of the present invention, the pH of the working fluid is 6.0 to 8.0.
In one embodiment of the invention, the pH of the working fluid is 7.3.
In one embodiment of the present invention, the pH of the working fluid is adjusted using an aqueous sodium hydroxide solution having a mass concentration of 8%.
The invention also provides the application of the method or the kit in biotin quantitative determination.
The technical scheme of the invention has the following advantages:
the invention provides a method for quantitatively determining biotin in a sample based on homogeneous phase time-resolved fluorescence, which comprises the enzyme digestion step, wherein the enzyme digestion step comprises the following steps: mixing the sample with protease, and performing enzymolysis to obtain an enzyme-digested sample; the method is based on homogeneous phase time-resolved fluorescence, and the homogeneous phase time-resolved fluorescence can be monitored by time resolution, so that a fluorescence signal can be easily distinguished from a short-life fluorescence background, and further background light interference is effectively avoided, and a quantitative determination result is more accurate; the method effectively avoids the difference of the biotinylation level of the protein and the deviation of the steric hindrance to the detection result of the immobilized biotin through enzyme digestion, and further improves the accuracy of the quantitative detection result; the detection Limit (LOD) of the method for quantitatively determining the biotin is only 0.03nM, and the determination sensitivity is extremely high; compared with the traditional ELISA method, the method does not need to clean unreacted reagents, greatly simplifies the steps of the quantitative determination of the biotin in the sample, and obviously shortens the period of the quantitative determination of the biotin in the sample by only 10-20 min in the determination process.
Further, the working reagent used in the method takes working solution as a solvent, and the components of the working solution comprise 4-hydroxyethyl piperazine ethanesulfonic acid, potassium fluoride and bovine serum albumin; the working solution can effectively maintain the ionic property and the pH value to be stable; the fluorine ions contained in the solution can react with Eu3+Forming a coordination, thereby improving the fluorescence signal; the contained bovine serum albumin can protect allophycocyanin in the working reagent, so that the activity of the reagent is in a long-term stable state.
Drawings
FIG. 1: and (3) taking the concentration of the biotin calibrator solution as an X axis and the signal value S as a Y axis, and drawing by four-parameter fitting to obtain a calibration curve.
FIG. 2: curve for biotin level determination in sample-biotin reagent premix mode in experimental example 2.
FIG. 3: assay curves for biotin levels in sample-avidin reagent premix mode in Experimental example 3.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1: kit for quantitatively determining biotin in sample
The present embodiment provides a kit for quantitative determination of biotin in a sample, the kit comprising:
proteinase K solution at a concentration of 10. mu.g/mL, proteinase K: purchased from Sigma, solvent: deionized water;
allophycocyanin-labeled biotin solution with biotin concentration of 12.5nM, allophycocyanin-labeled biotin taken from a biotin labeling kit, biotin labeling kit: purchased from Elabscience, solvent: a working fluid;
eu with streptavidin concentration of 2.5nM3+Labeled streptavidin solution, Eu3+Labeled streptavidin: purchased from thermo fisher Scientific, solvent: a working fluid;
wherein the components of the working solution comprise 11.916 g/L4-hydroxyethyl piperazine ethanesulfonic acid, 23.24g/L potassium fluoride, 2g/L bovine serum albumin, 0.5ml/L Lproclin300 and 0.5ml/L Tween 20, and the pH of the working solution is adjusted to 7.3 by using a sodium hydroxide aqueous solution with the mass concentration of 8%.
Example 2: method for quantitatively determining biotin in sample based on homogeneous phase time-resolved fluorescence
The embodiment provides a method for quantitatively determining biotin in a sample based on homogeneous time-resolved fluorescence, which comprises the following specific steps:
enzyme digestion step: mixing 150 mu L of sample with 1 mu L of proteinase K solution with the concentration of 10 mu g/mL in the embodiment 1, carrying out enzymolysis for 2h at 700rpm and 58 ℃, and then carrying out enzyme deactivation for 10min in 95 ℃ water bath to obtain an enzyme-digested sample;
an incubation step: firstly, 10 mu L of enzyme-digested sample is mixed with 5 mu L of allophycocyanin-labeled biotin solution with the biotin concentration of 12.5nM, then the mixture is incubated for 10min at 37 ℃ to obtain an incubation solution 1, and then the incubation solution 1 and 5 mu L of Eu with the streptavidin concentration of 2.5nM are mixed3+Mixing the marked streptavidin solution, and incubating for 10min at 37 ℃ to obtain an incubation liquid 2;
the determination step comprises: an infinite 200PRO microplate reader is used for exciting fluorescence of the incubation liquid 2 at an excitation wavelength of 320nm (bandwidth 25nm), and then fluorescence signals are detected at emission wavelengths of 620nm (bandwidth 20nm) and 665nm (bandwidth 8nm) (delay time 50 mus, integration time 400 mus);
a calculation step: substituting the measured fluorescence signal into a fitting equation obtained by fitting according to the relation between the fluorescence signal and the biotin concentration, and calculating to obtain the concentration of biotin in the sample; the fitting equation (R)20.994, LOD 0.03nM) as follows:
Y＝36311.3/[1+(x/5.398)^-1.395]+975.7；
in the fitting equation, x is the concentration of biotin in the sample, unit nM, and Y is the signal value S; the calculation formula of the signal value S is as follows:
S＝(λ665nm/λ620nm)^10000；
in the formula, λ665nmThe fluorescence signal of the incubation liquid 2 under the emission wavelength of 665nm is AU; lambda [ alpha ]620nmThe fluorescence signal of incubation fluid 2 at an emission wavelength of 620nm is given in AU.
The obtaining process of the fitting equation comprises the following steps:
configuration of the calibration product: preparing 11 biotin calibrator solutions with biotin concentrations of 100nM, 75nM, 50nM, 25nM, 10nM, 6nM, 3nM, 1nM, 0.2nM, 0.05nM and 0nM, respectively, using the working solution;
an incubation step: mixing 10 mu L of biotin calibrator solution with each concentration with 5 mu L of working reagent 1 marked by fluorescent group 1, incubating for 10min at 37 ℃ to obtain incubation liquid 1, mixing each incubation liquid 1 with 5 mu L of working reagent 2 marked by fluorescent group 2, and incubating for 10min at 37 ℃ to obtain incubation liquid 2;
the determination step comprises: fluorescence excitation was performed on each incubation liquid 2 by using an infinite 200PRO microplate reader at an excitation wavelength of 320nm (bandwidth 25nm), and then fluorescence signal detection was performed at emission wavelengths of 620nm (bandwidth 20nm) and 665nm (bandwidth 8nm), respectively (delay time 50 μ s, integration time 400 μ s);
fitting: collecting the fluorescence signal with the emission wavelength of 665nm and the fluorescence signal with the emission wavelength of 620nm measured by each incubation liquid 2, calculating 10000 times of the ratio of the fluorescence signal with the emission wavelength of 665nm to the fluorescence signal with the emission wavelength of 620nm, taking the ratio as a signal value S corresponding to the concentration of biotin in a sample, then drawing a calibration curve by four-parameter fitting by taking the concentration of a biotin calibrator solution as an X axis and the signal value S as a Y axis, and obtaining a fitting equation obtained by fitting according to the relation between the fluorescence signals and the biotin concentration.
Example 3: method for quantitatively determining biotin in sample based on homogeneous phase time-resolved fluorescence
This example provides a method for the quantitative determination of biotin in a sample based on homogeneous time-resolved fluorescence, which differs from example 2 in that:
an incubation step: firstly, 10 μ L of the enzyme-digested sample and 5 μ L of Eu having a streptavidin concentration of 2.5nM3+Mixing the labeled streptavidin solution, incubating at 37 ℃ for 10min to obtain an incubation solution 1, mixing the incubation solution 1 with 5 mu L of allophycocyanin labeled biotin solution with the biotin concentration of 12.5nM, and incubating at 37 ℃ for 10min to obtain an incubation solution 2.
Experimental example 1: quantitative determination of biotin in a sample
This example provides a quantitative assay of biotin in a sample, the procedure of which is as follows:
biotin-labeled antibodies in a kit of Roche electrochemiluminescence (PCT), AFP and CEA (purchased from Roche diagnostics products (Shanghai)) are selected as samples and are respectively marked as PCT-B, AFP-B, CEA-B, and the concentrations of the biotin-labeled antibodies provided by PCT-B, AFP-B, CEA-B are respectively 2.0mg/mL, 4.5mg/mL and 3.0 mg/mL.
The biotin concentration in PCT-B, AFP-B, CEA-B was quantitatively determined by the method described in example 2, using the enzyme-cleaved sample as a control. The results are shown in Table 1.
As can be seen from Table 1, in the sample-biotin reagent premixing mode, the result of measuring the biotin concentration increases by more than 38% compared with the non-digested sample after the digestion of the sample. The results indicate that the accuracy of the biotin quantitation was affected by the presence of biotin. Immobilized biotin in the labeled reagent product exists in a free form after enzyme digestion, the enzyme digestion effectively avoids the influence of the difference of the biotinylation level of protein and the steric hindrance between the immobilized biotin on the test result, and the accuracy of the quantitative determination result is further improved.
TABLE 1 concentration of biotin in PCT-B, AFP-B, CEA-B
Experimental example 2: quantitative determination of biotin in a sample
This example provides a quantitative assay of biotin in a sample, the procedure of which is as follows:
biotin-labeled bovine serum albumin (bovine serum albumin from sigma, biotin labeling kit from Elapscience, see the handbook of Biotin labeling kits) was selected as the sample (designated BSA-B), and labeling was performed at biotin/bovine serum albumin (B/BSA) ratios of 2:1, 4:1, 8:1, 12:1, 16:1, and 20:1 by volume (designated B2, B4, B8, B12, B16, and B20, respectively).
After the sample was diluted with the working solution of example 1 to a BSA concentration of 5nM, the biotin contents in B2, B4, B8, B12, B16 and B20 were each quantitatively measured by the method described in example 2. The results are shown in Table 2 and FIG. 2.
As is clear from Table 2 and FIG. 2, in the sample-biotin reagent premix mode, all the proteins to be labeled were controlled to be bovine serum albumin, but the biotin labeling ratio was different, the biotin concentration measurement results after enzyme digestion were increased relative to the samples without enzyme digestion, and the relative deviation thereof was increased with the increase in B/BSA and finally stabilized at about 20%. When the B/BSA is 4, the enzyme digestion result is very small compared with the enzyme digestion result. The results show that the increase of B/BSA leads to an increase of steric hindrance between immobilized biotin, resulting in more biotin which is not bound to avidin reagent, resulting in a lower level of immobilized biotin assay (not digested) than the actual biotin level (digested). It can be seen that, when the existence form of biotin and the biotinylation level of protein cannot be determined, the methods of example 1 and example 2 can effectively prevent the omission of biotin and improve the detection accuracy.
The biotin contents in tables 2B 2, B4, B8, B12, B16 and B20
Experimental example 3: quantitative determination of biotin in a sample
This example provides a quantitative assay of biotin in a sample, the procedure of which is as follows:
biotin-labeled bovine serum albumin (bovine serum albumin from sigma, biotin labeling kit from Elapscience, see the handbook of Biotin labeling kits) was selected as the sample (designated BSA-B), and labeling was performed at biotin/bovine serum albumin (B/BSA) ratios of 2:1, 4:1, 8:1, 12:1, 16:1, and 20:1 by volume (designated B2, B4, B8, B12, B16, and B20, respectively).
After the sample was diluted with the working solution of example 1 to a BSA concentration of 5nM, the biotin contents in B2, B4, B8, B12, B16 and B20 were each quantitatively measured by the method described in example 3. The results are shown in Table 3 and FIG. 3.
As can be seen from Table 3 and FIG. 3, in the sample-avidin reagent premixing mode, all the labeled proteins are controlled to be bovine serum albumin, but the labeling ratios of biotin are different, the biotin concentration measurement result after enzyme digestion is increased compared with the sample without enzyme digestion, the relative deviation is not more than 9%, and is reduced by about 11% compared with the sample-biotin reagent premixing mode. In addition, when the volume ratio of the biotin to the bovine serum albumin to be tested is within the range of 1-8, the relative deviation before and after enzyme digestion is not more than 2%. It can be seen that the sample-avidin reagent premixing mode in example 3 can significantly reduce the result deviation caused by enzyme digestion, and is more suitable for quantitative determination of immobilized biotin than the sample-biotin reagent premixing mode. And the biotinylation level of the protein is generally not more than 10 biotin/protein, so that the enzyme digestion step can be directly omitted during testing, and the test result can be quickly obtained.
The biotin contents in tables 3B 2, B4, B8, B12, B16 and B20
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for quantitatively determining biotin in a sample based on homogeneous phase time-resolved fluorescence is characterized by comprising an enzyme digestion step; the enzyme digestion step is as follows:
and mixing the sample with protease, and carrying out enzymolysis to obtain the enzyme-digested sample.
2. The method of claim 1, wherein the protease is a serine protease.
3. The method of claim 1 or 2, wherein the step of cleaving further comprises a step of incubation; the incubation steps are as follows:
firstly, mixing the enzyme-digested sample with a working reagent 1 marked by a fluorescent group 1, and then incubating to obtain an incubation liquid 1, and then mixing the incubation liquid 1 with a working reagent 2 marked by a fluorescent group 2, and then incubating to obtain an incubation liquid 2.
4. The method according to any one of claims 1 to 3, wherein the working reagent 1 labeled with the fluorophore 1 is allophycocyanin-labeled biotin and Eu3+At least one of labeled streptavidin.
5. The method according to claim 4, wherein when the working reagent 1 labeled with the fluorophore 1 is allophycocyanin-labeled biotin, the working reagent 2 labeled with the fluorophore 2 is Eu3+Labeled streptavidin;
when the working reagent 1 labeled with the fluorescent group 1 is Eu3+When streptavidin is marked, the working reagent 2 marked by the fluorescent group 2 is biotin marked by allophycocyanin.
6. The method of claim 4 or 5, wherein the allophycocyanin-labeled biotin solution and Eu3+The solvent of the marked streptavidin solution is working solution; the components of the working solution comprise a buffering agent, a salt and a protein protective agent.
7. The method of claim 6, wherein the working fluid comprises 2-60 g/L buffer, 5-100 g/L salt, and 0.4-10 g/L protein protectant.
8. The method of any one of claims 1 to 7, further comprising an assay step after the incubating step; the determination steps are as follows:
and (3) exciting the fluorescence of the incubation liquid 2, determining the fluorescence signal of the incubation liquid 2, and calculating to obtain the concentration of the biotin in the sample according to the fluorescence signal and the relation between the fluorescence signal and the concentration of the biotin.
9. A kit for quantitative determination of biotin in a sample, comprising a protease, a working reagent 1 labeled with a fluorophore 1, and a working reagent 2 labeled with a fluorophore 2.
10. Use of the method of any one of claims 1 to 8 or the kit of any one of claims 9 in a quantitative assay for biotin.
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