CN113533282B - 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 290
- 229960002685 biotin Drugs 0.000 title claims abstract description 150
- 239000011616 biotin Substances 0.000 title claims abstract description 150
- 235000020958 biotin Nutrition 0.000 title claims abstract description 145
- 238000004445 quantitative analysis Methods 0.000 title abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 36
- 238000002156 mixing Methods 0.000 claims abstract description 20
- 108091005804 Peptidases Proteins 0.000 claims abstract description 18
- 239000004365 Protease Substances 0.000 claims abstract description 18
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 11
- 238000002868 homogeneous time resolved fluorescence Methods 0.000 claims abstract description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 49
- 238000011534 incubation Methods 0.000 claims description 38
- 239000000243 solution Substances 0.000 claims description 32
- 108010090804 Streptavidin Proteins 0.000 claims description 31
- 239000012530 fluid Substances 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 28
- 108010004469 allophycocyanin Proteins 0.000 claims description 24
- 235000019419 proteases Nutrition 0.000 claims description 17
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 15
- 229940098773 bovine serum albumin Drugs 0.000 claims description 15
- 239000012224 working solution Substances 0.000 claims description 12
- NROKBHXJSPEDAR-UHFFFAOYSA-M potassium fluoride Chemical compound [F-].[K+] NROKBHXJSPEDAR-UHFFFAOYSA-M 0.000 claims description 10
- 239000002904 solvent Substances 0.000 claims description 10
- 108010067770 Endopeptidase K Proteins 0.000 claims description 7
- 239000011698 potassium fluoride Substances 0.000 claims description 5
- 235000003270 potassium fluoride Nutrition 0.000 claims description 5
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 229940089468 hydroxyethylpiperazine ethane sulfonic acid Drugs 0.000 claims description 2
- 238000005259 measurement Methods 0.000 abstract description 15
- 238000001514 detection method Methods 0.000 abstract description 11
- 230000006287 biotinylation Effects 0.000 abstract description 7
- 238000000338 in vitro Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 102000035195 Peptidases Human genes 0.000 description 13
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- 102000004169 proteins and genes Human genes 0.000 description 10
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- 150000003839 salts Chemical class 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 239000004094 surface-active agent Substances 0.000 description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 8
- 238000003018 immunoassay Methods 0.000 description 7
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 6
- 230000005284 excitation Effects 0.000 description 6
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- 102000004190 Enzymes Human genes 0.000 description 5
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- 238000003556 assay Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 229940088598 enzyme Drugs 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
- 230000008569 process Effects 0.000 description 5
- 239000007836 KH2PO4 Substances 0.000 description 4
- 108010022999 Serine Proteases Proteins 0.000 description 4
- 102000012479 Serine Proteases Human genes 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 238000007413 biotinylation Methods 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- 239000002736 nonionic surfactant Substances 0.000 description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 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
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- 108090000317 Chymotrypsin Proteins 0.000 description 2
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- 108010067372 Pancreatic elastase Proteins 0.000 description 2
- 102000016387 Pancreatic elastase Human genes 0.000 description 2
- 229920001214 Polysorbate 60 Polymers 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
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- 229910004613 CdTe Inorganic materials 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
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- 229910052700 potassium Inorganic materials 0.000 description 1
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- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
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Classifications
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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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- Health & Medical Sciences (AREA)
- 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 of 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 following steps: mixing the sample with protease for enzymolysis to obtain an enzyme-cut sample; the method is based on homogeneous time-resolved fluorescence, the homogeneous time-resolved fluorescence can be monitored through time resolution, fluorescent signals can be easily resolved from short-service-life fluorescent backgrounds, further, background light interference is effectively avoided, and quantitative measurement results are more accurate; the method effectively avoids the difference of protein biotinylation level and the deviation of steric hindrance on the immobilized biotin measurement result by enzyme digestion, and further improves the accuracy of the quantitative measurement result; the detection limit of the biotin quantitative determination method 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 of homogeneous phase time-resolved fluorescence.
Background
Biotin is a small molecule in the vitamin B group that 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, nanoparticles, and the like. The biological activity of these agents is hardly affected by biotin coupling. Furthermore, biotin has a highly selective and stable interaction with avidin and its homologs. The interaction of biotin with avidin is currently the strongest non-covalent interaction known, with an affinity constant as high as 10 15 mol/L. In addition to the usual streptavidin, more and more new streptavidin variants are designed to meet different experimental properties and requirements in vitro and in vivo studies. Meanwhile, various chemical and enzymatic biotinylation techniques based on biotin-streptavidin systems can be applied to various experiments, such as signal amplification and target isolation. In addition, the wide application of biotin as a marker of antibodies, nucleic acids, nanoparticles, etc. in the in vitro diagnostic industry makes the quantitative determination of biotin concentration of great potential value.
Currently, researchers have successfully studied ELISA (specific visual reference ：Yuo-Sheng Chang,C.-H.W.,Re-Jiin Chang,David Shiuan,Determination of biotin concentration by a competitive enzyme-linked immunosorbent assay(ELISA)method.J.Biochern.Biophys.Methods 1994,29,321-329.)、 chemiluminescence (specific visual reference ：Xing-guang,T.Z.S.a.S.,Determination ofBiotin in Pharmaceutical Formulations by Potassium Permanganate-luminol-CdTe Nanoparticles Chemiluminescence System.Chem.Res.Chinese Universities 2012,28(4),604-608.) and electrochemical competition (specific visual reference ：Liu,S.-L.;Chen,S.-Z.;Zhao,Q.;Xu,Z.-H.;Li,Y.;Jia,J.-H.;Guo,L.-H.,Photoelectrochemical Competitive Detection of Biotin.Chinese Journal ofAnalytical Chemistry 2013,41(10),1477-1481.) et al immunoassay-based methods of quantitative determination of biotin, measured limit of detection (LOD) and detection range can range from nanomole to micromolar levels).
However, with the increasing development of in vitro diagnostic techniques, the sensitivity and accuracy requirements of biotin quantitative determination are increasingly raised in many special scenes, and the immunoassay-based biotin quantitative determination methods such as ELISA, chemiluminescence and electrochemiluminescence techniques cannot meet the high requirements of these special scenes on the sensitivity and accuracy of biotin quantitative determination. For example, at present, immunoassay technology using biotin as a labeling molecule for antibodies, nucleic acids, quantum dots, nanoparticles, and the like occupies most of the market in the in vitro diagnostic industry. If the biotinylation level of the reagent can be accurately quantified in the early stage of the development of the biotin labeling diagnostic reagent, the development direction of the reagent can be effectively guided, and the development period is greatly shortened. Also, in biotin-streptavidin based immunoassays, biotin present in an unknown sample can compete with the biotin-labeled reagent, thereby interfering with the test results. As another example, biotin has been added to some over-the-counter drugs over the past few years and is used by more and more people for cosmetic needs such as hair, nails, and skin care. Frequent contact with or consumption of the biotin care product may indirectly or directly cause an increase in the concentration of biotin in the blood, resulting in misdiagnosis of clinical blood analysis. Furthermore, biotin itself is an indispensable vitamin involved in metabolism in humans, and the concentration of biotin in normal human plasma is not more than 2.05nM. Accurate monitoring of the biotin level in the blood plasma can effectively screen genetic metabolic diseases lacking biotin and guide patients to take the biotin reasonably. The above-described special scenarios all require high sensitivity and high precision detection of samples with lower biotin levels to determine biotin interference in biotin-streptavidin based immunoassays, as well as to guide in vitro diagnostic reagent development and clinical biotin therapy.
In addition, the immunoassay-based quantitative determination methods of biotin such as ELISA, chemiluminescence and electrochemiluminescence techniques have some drawbacks. For example, ELISA requires an enzyme substrate and additional washing, with a measurement period of up to several hours. For example, the quantitative measurement methods of biotin by immunoassay such as ELISA, chemiluminescence and electrochemiluminescence techniques are all detection based on an instant signal, and are easily interfered by self-luminescence of reagents, substrates and solvents, and the result is deviated.
Therefore, it is highly desirable to find a quantitative biotin assay method which has high sensitivity, good accuracy, simple steps and short assay 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 performing enzymolysis to obtain an 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 present invention, the protease is a protease solution having a concentration of 50 to 100. Mu.g/mL.
In one embodiment of the invention, the solvent of the protease solution is at least one of water, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), tris (hydroxymethyl) aminomethane (Tris), naH 2PO4&Na2HPO4 and K 2HPO4&KH2PO4.
In one embodiment of the invention, the enzymatic hydrolysis is: enzymolysis is carried out for 15-120 min at 55-65 ℃.
In one embodiment of the invention, the enzymatic hydrolysis is: and (3) performing enzymolysis for 2 hours 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 sample to protease mixing volume ratio is 150:1.
In one embodiment of the invention, the step of cleaving further comprises an incubation step; the incubation steps are as follows:
Firstly, mixing an enzyme-digested sample with a working reagent 1 marked by a fluorescent group 1, 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, incubating to obtain an incubation liquid 2.
In one embodiment of the present invention, the working reagent 1 labeled with the fluorescent group 1 is at least one of allophycocyanin-labeled biotin and Eu 3+ -labeled streptavidin.
In one embodiment of the present invention, when the working reagent 1 labeled with the fluorescent group 1 is allophycocyanin-labeled biotin, the working reagent 2 labeled with the fluorescent group 2 is Eu 3+ -labeled streptavidin;
when the working reagent 1 labeled with the fluorescent group 1 is streptavidin labeled with Eu 3+, the working reagent 2 labeled with the fluorescent group 2 is biotin labeled with allophycocyanin.
In one embodiment of the invention, the allophycocyanin-labeled biotin is a solution of allophycocyanin-labeled biotin having a biotin concentration of 1 to 20 nM.
In one embodiment of the invention, the allophycocyanin-labeled biotin is a solution of allophycocyanin-labeled biotin having a biotin concentration of 12.5 nM.
In one embodiment of the present invention, the Eu 3+ -labeled streptavidin is Eu 3+ -labeled streptavidin solution having a concentration of 0.2-4 nM of streptavidin.
In one embodiment of the present invention, the Eu 3+ -labeled streptavidin is Eu 3+ -labeled streptavidin solution with a streptavidin concentration of 2.5 nM.
In one embodiment of the present invention, the solvents of the allophycocyanin-labeled biotin solution and the Eu 3+ -labeled streptavidin solution are working solutions; the components of the working fluid comprise a buffering agent, a salt and a protein protecting agent.
In one embodiment of the invention, the buffer is at least one of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), tris (hydroxymethyl) aminomethane (Tris), naH 2PO4&Na2HPO4, and K 2HPO4&KH2PO4; the salt is potassium fluoride; the protein protectant is bovine serum albumin.
In one embodiment of the present invention, the working fluid comprises 2 to 60g/L buffer, 5 to 100g/L salt, and 0.4 to 10g/L protein protectant.
In one embodiment of the present invention, the working fluid comprises 5 to 15g/L buffer, 10 to 30g/L salt and 1 to 3g/L protein protectant.
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 components of the working fluid further comprise 0.1 to 1ml/L of a surfactant.
In one embodiment of the invention, the composition of the working fluid further comprises 0.5ml/L surfactant.
In one embodiment of the invention, the surfactant is a nonionic surfactant.
In one embodiment of the present invention, the nonionic surfactant is at least one of tween 20, tween 40, tween 60, tween 80, triton 100 and triton 114.
In one embodiment of the invention, the components of the working fluid further comprise a preservative.
In one embodiment of the present invention, the components of the working fluid further comprise 0.1 to 1ml/L of a preservative.
In one embodiment of the invention, the composition of the working fluid further comprises 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 invention, the pH of the working fluid is adjusted using an 8% strength by mass aqueous sodium hydroxide solution.
In one embodiment of the invention, the incubation is: incubating at 25-43 ℃ for 5-30 min.
In one embodiment of the invention, the incubation is: incubate at 37℃for 10min.
In one embodiment of the invention, the mixing volume ratio of the 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 digested sample to the working reagent 1 labeled with the fluorescent group 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 invention, the mixing volume ratio of the incubation liquid 1 to the working agent 2 labeled with the fluorescent group 2 is 3:1.
In one embodiment of the invention, the incubating step is followed by an assay step; the measuring steps are as follows:
And (3) performing fluorescence excitation on the incubation liquid 2, determining a fluorescence signal of the incubation liquid 2, and finally calculating the concentration of biotin in the sample according to the fluorescence signal and the relationship between the fluorescence signal and the concentration of biotin.
In one embodiment of the invention, the excitation wavelength of the fluorescent signal is 320nm.
In one embodiment of the invention, the emission wavelength of the fluorescent signal is 620nm and 665nm.
In one embodiment of the invention, the calculation is: bringing the fluorescence signal into a fitting equation obtained by fitting according to the relationship 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 a sample, the unit is nM, and Y is a signal value S; the calculation formula of the signal value S is as follows:
S＝(λ665nm/λ620nm)^10000；
In the formula, lambda 665nm is a fluorescent signal of the incubation liquid 2 under the emission wavelength of 665nm, and the unit is AU; lambda 620nm is the fluorescence signal of incubation liquid 2 at an emission wavelength of 620nm, in AU.
The invention also provides a kit for quantitative determination of biotin in a sample, which comprises protease, a working reagent 1 labeled with a fluorescent group 1 and a working reagent 2 labeled with a 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 present invention, the protease is a protease solution having a concentration of 50 to 100. Mu.g/mL.
In one embodiment of the invention, the solvent of the protease solution is at least one of water, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), tris (hydroxymethyl) aminomethane (Tris), naH 2PO4&Na2HPO4 and K 2HPO4&KH2PO4.
In one embodiment of the present invention, the working reagent 1 labeled with the fluorescent group 1 is at least one of allophycocyanin-labeled biotin and Eu 3+ -labeled streptavidin.
In one embodiment of the present invention, when the working reagent 1 labeled with the fluorescent group 1 is allophycocyanin-labeled biotin, the working reagent 2 labeled with the fluorescent group 2 is Eu 3+ -labeled streptavidin;
when the working reagent 1 labeled with the fluorescent group 1 is streptavidin labeled with Eu 3+, the working reagent 2 labeled with the fluorescent group 2 is biotin labeled with allophycocyanin.
In one embodiment of the invention, the allophycocyanin-labeled biotin is a solution of allophycocyanin-labeled biotin having a biotin concentration of 1 to 20 nM.
In one embodiment of the invention, the allophycocyanin-labeled biotin is a solution of allophycocyanin-labeled biotin having a biotin concentration of 12.5 nM.
In one embodiment of the present invention, the Eu 3+ -labeled streptavidin is Eu 3+ -labeled streptavidin solution having a concentration of 0.2-4 nM of streptavidin.
In one embodiment of the present invention, the Eu 3+ -labeled streptavidin is Eu 3+ -labeled streptavidin solution with a streptavidin concentration of 2.5 nM.
In one embodiment of the present invention, the solvents of the allophycocyanin-labeled biotin solution and the Eu 3+ -labeled streptavidin solution are working solutions; the components of the working fluid comprise a buffering agent, a salt and a protein protecting agent.
In one embodiment of the invention, the buffer is at least one of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), tris (hydroxymethyl) aminomethane (Tris), naH 2PO4&Na2HPO4, and K 2HPO4&KH2PO4; the salt is potassium fluoride; the protein protectant is bovine serum albumin.
In one embodiment of the present invention, the working fluid comprises 2 to 60g/L buffer, 5 to 100g/L salt, and 0.4 to 10g/L protein protectant.
In one embodiment of the present invention, the working fluid comprises 5 to 15g/L buffer, 10 to 30g/L salt and 1 to 3g/L protein protectant.
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 components of the working fluid further comprise 0.1 to 1ml/L of a surfactant.
In one embodiment of the invention, the composition of the working fluid further comprises 0.5ml/L surfactant.
In one embodiment of the invention, the surfactant is a nonionic surfactant.
In one embodiment of the present invention, the nonionic surfactant is at least one of tween 20, tween 40, tween 60, tween 80, triton 100 and triton 114.
In one embodiment of the invention, the components of the working fluid further comprise a preservative.
In one embodiment of the present invention, the components of the working fluid further comprise 0.1 to 1ml/L of a preservative.
In one embodiment of the invention, the composition of the working fluid further comprises 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 invention, the pH of the working fluid is adjusted using an 8% strength by mass aqueous sodium hydroxide solution.
The invention also provides an application of the method or the kit in quantitative determination of biotin.
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 following steps: mixing the sample with protease for enzymolysis to obtain an enzyme-cut sample; the method is based on homogeneous time-resolved fluorescence, the homogeneous time-resolved fluorescence can be monitored through time resolution, fluorescent signals can be easily resolved from short-service-life fluorescent backgrounds, further, background light interference is effectively avoided, and quantitative measurement results are more accurate; the method effectively avoids the difference of protein biotinylation level and the deviation of steric hindrance on the immobilized biotin measurement result by enzyme digestion, and further improves the accuracy of the quantitative measurement result; the detection Limit (LOD) of the biotin quantitatively measured by the method is only 0.03nM, and the measurement sensitivity is extremely high; compared with the traditional ELISA method, the method does not need to clean unreacted reagent, greatly simplifies the step of quantitative determination of biotin in a sample, and simultaneously, the determination process only needs 10-20 min, thereby obviously shortening the period of quantitative determination of biotin in the sample.
Further, the working reagent used in the method takes working solution as solvent, and the components of the working solution comprise 4-hydroxyethyl piperazine ethane sulfonic acid, potassium fluoride and bovine serum albumin; the working solution can effectively maintain the ion characteristic and the pH value stable; the fluorine ions can form coordination with Eu 3+, so that fluorescence signals are improved; the 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) drawing a calibration curve by four-parameter fitting by taking the concentration of the biotin calibrator solution as an X axis and the signal value S as a Y axis.
Fig. 2: measurement curve of biotin level in sample-biotin reagent premix mode in experimental example 2.
Fig. 3: assay profile of biotin levels in sample-avidin reagent premix mode in experimental example 3.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1: kit for quantitatively determining biotin in sample
The present embodiment provides a kit for quantitatively determining 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;
An allophycocyanin-labeled biotin solution having a biotin concentration of 12.5nM, allophycocyanin-labeled biotin being taken from a biotin labeling kit, biotin labeling kit: purchased from Elabscience company, solvent: a working fluid;
Eu 3+ labeled streptavidin solution with streptavidin concentration of 2.5nM, eu 3+ labeled streptavidin: purchased from ThermoFisher Scientific company, solvent: a working fluid;
Wherein the working solution comprises 11.916 g/L4-hydroxyethyl piperazine ethane sulfonic acid, 23.24g/L potassium fluoride, 2g/L bovine serum albumin, 0.5ml/Lproclin300 and 0.5ml/L Tween 20, and the pH of the working solution is regulated 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 phase time-resolved fluorescence, which comprises the following specific steps:
and (3) enzyme cutting: mixing 150 μl of sample with 1 μl of proteinase K solution of example 1 with concentration of 10 μg/mL, performing enzymolysis at 700rpm and 58 deg.C for 2 hr, and inactivating enzyme in 95deg.C water bath for 10min to obtain digested sample;
Incubation: firstly, mixing 10 mu L of a sample subjected to enzyme digestion with 5 mu L of allophycocyanin marked biotin solution with the biotin concentration of 12.5nM, incubating at 37 ℃ for 10min to obtain an incubation liquid 1, then mixing the incubation liquid 1 with 5 mu L of Eu 3+ marked streptavidin solution with the streptavidin concentration of 2.5nM, and incubating at 37 ℃ for 10min to obtain an incubation liquid 2;
The measuring step comprises the following steps: excitation of fluorescence is carried out on the incubation liquid 2 by using an infinite 200PRO enzyme-labeled instrument at an excitation wavelength of 320nm (bandwidth of 25 nm), and then detection of fluorescence signals is carried out at emission wavelengths of 620nm (bandwidth of 20 nm) and 665nm (bandwidth of 8 nm) respectively (delay time of 50 mu s and integration time of 400 mu s);
the calculation steps are as follows: carrying 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 the biotin in the sample; the fit equation (R 2 is 0.994, LOD is 0.03 nM) 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 a sample, the unit is nM, and Y is a signal value S; the calculation formula of the signal value S is as follows:
S＝(λ665nm/λ620nm)^10000；
In the formula, lambda 665nm is a fluorescent signal of the incubation liquid 2 under the emission wavelength of 665nm, and the unit is AU; lambda 620nm is the fluorescence signal of incubation liquid 2 at an emission wavelength of 620nm, in AU.
The fitting equation obtaining process comprises the following steps:
Configuration of the calibrator: working solutions were used to prepare 11 total biotin calibrator solutions at biotin concentrations of 100nM, 75nM, 50nM, 25nM, 10nM, 6nM, 3nM, 1nM, 0.2nM, 0.05nM, 0nM, respectively;
Incubation: firstly, 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, then 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 measuring step comprises the following steps: excitation of fluorescence is carried out on each incubation liquid 2 by using an infinite 200PRO enzyme-labeled instrument before excitation wavelength of 320nm (bandwidth of 25 nm), and then detection of fluorescence signals is carried out at emission wavelengths of 620nm (bandwidth of 20 nm) and 665nm (bandwidth of 8 nm) respectively (delay time of 50 mu s and integration time of 400 mu s);
Fitting: firstly collecting fluorescence signals with emission wavelength of 665nm and fluorescence signals with emission wavelength of 620nm measured by each incubation liquid 2, calculating 10000 times of the ratio of the fluorescence signals with emission wavelength of 665nm to the fluorescence signals with emission wavelength of 620nm, taking the value as a signal value S corresponding to the concentration of biotin in a sample, then taking the concentration of biotin calibrator solution as an X axis and the signal value S as a Y axis, and drawing a calibration curve through four-parameter fitting to obtain a fitting equation obtained through fitting according to the relationship between the fluorescence signals and the concentration of biotin.
Example 3: method for quantitatively determining biotin in sample based on homogeneous phase time-resolved fluorescence
This example provides a method for quantitative determination of biotin in a sample based on homogeneous time-resolved fluorescence, which differs from example 2 in that:
Incubation: firstly, 10 mu L of a sample subjected to enzyme digestion is mixed with 5 mu L of Eu 3+ marked streptavidin solution with the concentration of 2.5nM, then incubated for 10min at 37 ℃ to obtain an incubation liquid 1, and then, after mixing the incubation liquid 1 with 5 mu L of allophycocyanin marked biotin solution with the concentration of 12.5nM, incubation is carried out for 10min at 37 ℃ to obtain an incubation liquid 2.
Experimental example 1: quantitative determination of biotin in a sample
The experimental example provides a quantitative determination experiment of biotin in a sample, and the experimental process is as follows:
Biotin-labeled antibodies in the Roche electrochemiluminescence PCT, AFP, CEA kit (available from Roche diagnostics products (Shanghai)) were selected as samples, and were recorded as PCT-B, AFP-B, CEA-B, respectively, and PCT-B, AFP-B, CEA-B provided biotin-labeled antibodies at concentrations of 2.0mg/mL, 4.5mg/mL, and 3.0mg/mL, respectively.
The biotin concentration in PCT-B, AFP-B, CEA-B was quantitatively determined using the method described in example 2, respectively, using the non-digested sample as a control. The measurement results are shown in Table 1.
As is clear from Table 1, in the sample-biotin reagent premixing mode, the biotin concentration measurement result increased by 38% or more after the sample was digested, compared with the undigested sample. The results demonstrate that the accuracy of the biotin quantitative determination is affected by the presence of biotin. The immobilized biotin in the labeled reagent product exists in a free form after enzyme digestion, so that the enzyme digestion effectively avoids the difference of protein biotinylation levels and the influence of steric hindrance among the immobilized biotin on a test result, and further improves the accuracy of quantitative determination results.
Table 1 PCT-B, AFP-B, CEA-B concentration of biotin
Experimental example 2: quantitative determination of biotin in a sample
The experimental example provides a quantitative determination experiment of biotin in a sample, and the experimental process is as follows:
Biotin-labeled bovine serum albumin (bovine serum albumin is purchased from sigma, biotin labeling kit is purchased from Elabscience, laboratory manual of biotin labeling kit) was selected as sample (labeled BSA-B), and labeling was performed at ratios of biotin/bovine serum albumin (B/BSA) of 2:1, 4:1, 8:1, 12:1, 16:1, and 20:1 by volume, respectively (labeled 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 content in B2, B4, B8, B12, B16 and B20 was quantitatively determined, respectively, by the method described in example 2. The measurement results are shown in Table 2 and FIG. 2.
As is clear from table 2 and fig. 2, in the sample-biotin reagent premixing mode, the labeled proteins were all controlled to be bovine serum albumin, and the biotin labeling ratio was different, so that the biotin concentration measurement result after digestion was increased relative to that of the non-digested sample, and the relative deviation was increased with the increase of B/BSA, and finally stabilized at about 20%. At B/BSA of 4, the cleavage and non-cleavage results differ slightly. The results demonstrate that an increase in B/BSA increases the steric hindrance between immobilized biotin, resulting in the presence of more biotin that fails to bind to the avidin reagent, resulting in an immobilized biotin assay (uncleaved) that is lower than the actual biotin level (cleaved). It can be seen that, when the presence form of biotin and the biotinylation level of protein were not determined, the methods of example 1 and example 2 were employed, which effectively prevented missed detection of biotin and improved detection accuracy.
Biotin content in tables 2 B2, B4, B8, B12, B16 and B20
Experimental example 3: quantitative determination of biotin in a sample
The experimental example provides a quantitative determination experiment of biotin in a sample, and the experimental process is as follows:
Biotin-labeled bovine serum albumin (bovine serum albumin is purchased from sigma, biotin labeling kit is purchased from Elabscience, laboratory manual of biotin labeling kit) was selected as sample (labeled BSA-B), and labeling was performed at ratios of biotin/bovine serum albumin (B/BSA) of 2:1, 4:1, 8:1, 12:1, 16:1, and 20:1 by volume, respectively (labeled 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 content in B2, B4, B8, B12, B16 and B20 was quantitatively determined, respectively, by the method described in example 3. The measurement 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, the labeled proteins were controlled to be bovine serum albumin, and the biotin labeling ratio was different, so that the biotin concentration measurement result after digestion was increased by not more than 9% relative to that of the non-digested sample, and the relative deviation was reduced by about 11% as compared with the sample-biotin reagent premixing mode. In addition, when the volume ratio of biotin/bovine serum albumin tested was in the range of 1 to 8, the relative deviation before and after cleavage was not more than 2%. It can be seen that the sample-avidin reagent premixing mode in example 3 can significantly reduce the deviation of results due to 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 the test, and the test result can be obtained rapidly.
Biotin content in tables 3 B2, B4, B8, B12, B16 and B20
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (4)
1. A method for quantitatively determining biotin in a sample based on homogeneous phase time-resolved fluorescence is characterized by sequentially comprising an enzyme digestion step, an incubation step and a determination step;
The enzyme digestion step is as follows: mixing the sample with protease for enzymolysis to obtain an enzyme-cut sample;
The incubation steps are as follows: firstly, mixing an enzyme-cut sample with a working reagent 1 marked by a fluorescent group 1, 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, incubating to obtain an incubation liquid 2;
The measuring steps are as follows: firstly, exciting fluorescence of the incubation liquid 2, then determining a fluorescence signal of the incubation liquid 2, and finally, calculating the concentration of biotin in a sample according to the relationship between the fluorescence signal and the concentration of biotin;
The working reagent 1 marked by the fluorescent group 1 is streptavidin marked by Eu 3+; the working reagent 2 marked by the fluorescent group 2 is allophycocyanin marked biotin;
the protease is proteinase K.
2. The method of claim 1, wherein the solvents of the allophycocyanin-labeled biotin solution and the Eu 3+ -labeled streptavidin solution are working fluids; the working solution comprises 11.916 g/L4-hydroxyethyl piperazine ethane sulfonic acid, 23.24g/L potassium fluoride, 2g/L bovine serum albumin, 0.5ml/Lproclin and 0.5ml/L tween 20; the pH of the working solution is 7.3.
3. A kit for quantitative determination of biotin in a sample, characterized in that the kit employs the method for quantitative determination of biotin in a sample based on homogeneous time-resolved fluorescence according to claim 1 or 2, comprising protease, working reagent 1 labeled with fluorescent group 1 and working reagent 2 labeled with fluorescent group 2; the working reagent 1 marked by the fluorescent group 1 is streptavidin marked by Eu 3+; the working reagent 2 marked by the fluorescent group 2 is allophycocyanin marked biotin; the protease is proteinase K.
4. Use of the kit of claim 3 in a quantitative determination of biotin.
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| EP0762122A1 (en) * | 1995-08-16 | 1997-03-12 | Bayer Ag | Streptavadin and biotin-based optical solid-phase biosensor |
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| US20050208598A1 (en) * | 2004-02-13 | 2005-09-22 | Cox W G | Biotin recognition sensors and high-throughput assays |
| WO2009105583A1 (en) * | 2008-02-19 | 2009-08-27 | Anstron Technologies Company | Fluorescence resonance energy transfer (fret) binding assays that are surface-based |
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| EP0762122A1 (en) * | 1995-08-16 | 1997-03-12 | Bayer Ag | Streptavadin and biotin-based optical solid-phase biosensor |
| WO2020033539A1 (en) * | 2018-08-10 | 2020-02-13 | Qoolabs, Inc. | Lateral flow assay for assessing target conjugation |
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| A study on the detection of free and bound biotin based on TR-FRET technology;Heng Chen 等;《Analyst》(第147期);318-324 * |
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