CN108801961B - Biotin detection method - Google Patents
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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 158
- 239000011616 biotin Substances 0.000 title claims abstract description 80
- 229960002685 biotin Drugs 0.000 title claims abstract description 80
- 235000020958 biotin Nutrition 0.000 title claims abstract description 79
- 238000001514 detection method Methods 0.000 title abstract description 18
- 238000002835 absorbance Methods 0.000 claims abstract description 32
- 239000007853 buffer solution Substances 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 22
- 150000001875 compounds Chemical class 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims abstract description 17
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims abstract description 9
- 239000002253 acid Substances 0.000 claims abstract description 8
- 238000000862 absorption spectrum Methods 0.000 claims description 14
- 230000008859 change Effects 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- 239000002563 ionic surfactant Substances 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 7
- 230000004151 fermentation Effects 0.000 abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 6
- 108090001008 Avidin Proteins 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 4
- 108010090804 Streptavidin Proteins 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000001228 spectrum Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000013048 microbiological method Methods 0.000 description 2
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- 229910001414 potassium ion Inorganic materials 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- 238000004611 spectroscopical analysis Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
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- 238000003556 assay Methods 0.000 description 1
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- 239000003093 cationic surfactant Substances 0.000 description 1
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- 230000036541 health Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
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- 230000009870 specific binding Effects 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Investigating Or Analysing Materials By Optical Means (AREA)
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Abstract
The invention discloses a Chinese yamA method for detecting a substance, comprising the steps of: a. get CH3(CH2)15(CH3)3N‑AuCl4Adding the compound into PBS buffer solution, adding standard biotin to form standard biotin solutions with different concentrations, uniformly mixing, adding into a cuvette, recording the absorbance value at the 267nm wavelength, and drawing a standard curve; b. get CH3(CH2)15(CH3)3N‑AuCl4And adding the compound into PBS buffer solution, adding the biotin sample to be detected, uniformly mixing, adding into a cuvette, recording the absorbance at the wavelength of 267nm, and calculating to obtain the concentration of the biotin sample to be detected through a standard working curve. The detection method directly utilizes the complex of chloroauric acid and CTAB, does not need a complex synthesis process, and is simple to operate; without resorting to the use of expensive reagents such as avidin or streptavidin; expensive analytical instruments are not required; the spectrum in the ultraviolet interval is used for analysis, so that the interference caused by the color of the fermentation liquor is avoided.
Description
Technical Field
The invention relates to a biotin detection method, in particular to a method capable of quickly, accurately and sensitively detecting biotin, and belongs to the field of biosensing.
Background
Biotin, as a vitamin in the B group, participates in various metabolic activities of the body, is widely applied to the fields of food, medicine, feed, fermentation and the like, and has important influence on industrial and agricultural production and daily life of people. The industrial and agricultural production related to biotin can be guided by detecting the content of biotin, so that the optimization of products is realized, and the health of people is maintained. At present, the detection methods of biotin at home and abroad mainly comprise a microbiological method, a high performance liquid chromatography, an enzyme-linked immunoassay method, a spectroscopic method, an electrochemical method and the like. However, the microbiological method has poor accuracy and the whole operation process is time-consuming and labor-consuming; instruments and reagents required by the high performance liquid chromatography are expensive, the sample processing process is complex, and the technical requirements on research personnel are high; although enzyme-linked immunosorbent assay and spectroscopy based on specific binding of biotin and avidin have the advantages of simple operation, high analysis speed and high accuracy, the reagent avidin or streptavidin used in the method has high market cost and is difficult to popularize and apply in production. Therefore, it is of great significance to develop a cheap, simple, fast, accurate and sensitive biotin detection method.
Disclosure of Invention
Aiming at the prior art, the invention provides a quick, accurate, sensitive and low-cost biotin detection method.
The invention is realized by the following technical scheme:
a biotin detection method comprises the following steps:
a method for detecting biotin, comprising: the method comprises the following steps:
a. taking CH of 0.1-2 m L3(CH2)15(CH3)3N-AuCl4Adding the compound into 3M L0.01.01M PBS buffer solution, adding standard biotin to form standard biotin solutions with different concentrations, uniformly mixing, adding the standard biotin solutions into a cuvette, recording the absorbance values of the standard biotin solutions with different concentrations at the 267nm wavelength, and drawing a standard curve by using the change value of the absorbance A at the 267nm wavelength and the concentration of the biotin;
b. taking CH of 0.1-2 m L3(CH2)15(CH3)3N-AuCl4Adding the complex into 3M L0.01.01M PBS buffer solution, adding biotin sample to be detected, mixing, adding into cuvette, recording absorbance at 267nm, and passing standardAnd working curve, and calculating to obtain the concentration of the biotin sample to be detected.
Or: the method comprises the following steps:
a. taking CH of 0.1-2 m L3(CH2)15(CH3)3N-AuCl4Adding the compound into 3M L0.01.01M PBS buffer solution, uniformly mixing, and adding into a cuvette to obtain an absorption spectrum with a wavelength range of 500-200 nm as a baseline;
b. taking CH of 0.1-2 m L3(CH2)15(CH3)3N-AuCl4Adding the compound into 3M L0.01.01M PBS buffer solution, adding standard biotin to form standard biotin solutions with different concentrations, mixing uniformly, and adding into a cuvette to obtain absorption spectra of biotin with different concentrations;
c. recording the absorbance values of standard biotin solutions with different concentrations at the 267nm wavelength, and drawing a standard curve by using the change value of the absorbance A at the 267nm wavelength and the concentration of biotin;
d. taking CH of 0.1-2 m L3(CH2)15(CH3)3N-AuCl4Adding the compound into 3M L0.01.01M PBS buffer solution, adding a biotin sample to be detected, uniformly mixing, adding into a cuvette to obtain an absorption spectrum with a wavelength range of 500-200 nm, recording the absorbance at the 267nm wavelength, and calculating by a standard working curve and a conventional method to obtain the concentration of the biotin sample to be detected.
The CH3(CH2)15(CH3)3N-AuCl4The compound is obtained by reacting chloroauric acid with cationic surfactant Cetyl Trimethyl Ammonium Bromide (CTAB), wherein 10-1000 mg of CTAB is taken, 50ml of CTAB is added with water, heated, stirred and dissolved, chloroauric acid solution with the concentration of 0.01-5 mg/L is added at room temperature, and stirring is carried out for 0.5-10 hours to obtain orange CH3(CH2)15(CH3)3N-AuCl4And (c) a complex.
The detection principle of the biotin detection method of the invention is as follows: chloroauric acid and hexadecyl trimethyl bromideAmmonium may form CH3(CH2)15(CH3)3N-AuCl4The compound, added with biotin sample can replace AuCl4 -Form CH3(CH2)15(CH3)3N-biotin, which results in a change in the absorbance spectrum and thus in a change in absorbance. And measuring the change value of absorbance (the absorbance value at the 267nm wavelength) after adding different concentrations of biotin by using an ultraviolet-visible spectrophotometer, drawing a standard working curve by using the change value of the absorbance A at the 267nm wavelength to the concentration C of the biotin, and contrasting the standard working curve to obtain the concentration of the biotin in the unknown sample.
The biotin detection method has the following advantages:
1. the invention directly utilizes the complex of chloroauric acid and CTAB, and does not need a complex synthesis process, thereby having simple operation and small error.
2. The invention does not need to use expensive reagents such as avidin or streptavidin, thereby greatly saving the detection cost.
3. The invention does not need expensive analytical instruments, and has the advantages of simple method, high detection speed, high sensitivity and wide linear range.
4. The invention utilizes the spectrum of the ultraviolet interval to analyze, and avoids the interference caused by the color of the fermentation liquor.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: CH with varying amounts of CTAB3(CH2)15(CH3)3N-AuCl4Absorption spectrum of the composite.
FIG. 2: response curves for different concentrations of biotin.
FIG. 3: standard working curve for biotin (low concentration range on left and high concentration range on right).
FIG. 4: results of the selectivity test of the detection method of the invention.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1 testing of the concentration of Biotin in a buffer solution
The determination steps are as follows:
a. with CH3(CH2)15(CH3)3N-AuCl4The complex is a recognition molecule, 0.01M PBS is a buffer solution, an ultraviolet-visible spectrophotometer is used for recording an absorption spectrum, and 0.5M L CH is taken3(CH2)15(CH3)3N-AuCl4And adding the compound into 3M L0.01.01M PBS buffer solution, uniformly mixing, adding into a cuvette, obtaining an absorption spectrum (shown in figure 1) in a wavelength range of 500-200 nm, and recording the absorbance at 267nm as a baseline.
CH3(CH2)15(CH3)3N-AuCl4The preparation process of the compound comprises the steps of taking 50mg CTAB, adding 50ml of water, heating, stirring and dissolving, adding 0.1 mg/L chloroauric acid solution at room temperature, and stirring for 1 hour to obtain orange CH3(CH2)15(CH3)3N-AuCl4And (c) a complex.
b. Take CH of 0.5m L3(CH2)15(CH3)3N-AuCl4The composition of the composite is prepared by mixing the components,adding the mixture into 3M L0.01.01M PBS buffer solution, adding biotin to obtain standard biotin solutions (5-400 mu g/L) with different concentrations, uniformly mixing, and adding the mixture into a cuvette to obtain absorption spectra of the biotin with different concentrations, wherein the absorption spectra are shown in figure 2.
c. The absorbance values at 267nm were recorded, respectively, and a standard curve was plotted with the change in absorbance A at 267nm and the concentration of biotin, as shown in FIG. 3. The standard curve can also be calculated from the curves of step a and step b.
And (3) performance testing: the interference of the method on glucose, lactose, maltose, sucrose, sodium ions, potassium ions, magnesium ions, chloride ions and the like is measured.
Take CH of 0.5m L3(CH2)15(CH3)3N-AuCl4The compound is added into PBS buffer solution with the concentration of 3M L0.01.01M, then biotin, glucose, lactose, maltose, sucrose, sodium ions, potassium ions, magnesium ions and chloride ions are respectively added to make the final concentrations of the biotin, the glucose, the lactose and the chloride ions all be 100 mu g/L, the mixture is respectively added into a cuvette after being uniformly mixed to obtain the absorption spectra of the objects to be detected, as shown in figure 4, the figure 4 shows that the absorbance of the biotin with the concentration of 100 mu g/L at 267nm is larger, and the absorbance of the glucose, the lactose, the maltose and the like at 267nm is small and can be ignored, so the ions can not influence the detection of the biotin, and the method has better selectivity, and is expected to be applied to the detection of the biotin in fermentation liquor or cell liquid.
Example 2
Two spiked samples were prepared from 0.01M PBS buffer at 20. mu.g/L and 200. mu.g/L, respectively, and the absorbance A at 267nm was recorded according to the assay of example 1 and the corresponding concentrations were calculated against a standard working curve (see FIG. 3).
Example 3 testing of the content of Biotin in the feed
The determination steps are as follows:
a. 1g of a feed was dissolved in 100ml of 0.01M PBS buffer solution, and insoluble matter was removed by filtration.
b. Take CH of 0.5m L3(CH2)15(CH3)3N-AuCl4And adding the compound into 3M L0.01.01M PBS buffer solution, adding a feed sample containing biotin, uniformly mixing, adding into a cuvette to obtain an absorption spectrum within the wavelength range of 500-200 nm, recording the absorbance at the 267nm wavelength, and obtaining the content of the biotin in the feed to be detected by comparing the absorbance A at the 267nm wavelength of the sample solution with a standard working curve.
Example 4 testing of the fermentation broth for Biotin content
The determination steps are as follows:
a. taking a certain fermentation liquid, adding 0.01M PBS buffer solution to dilute by 10 times, filtering to remove insoluble substances, and standing for testing.
b. Take CH of 0.5m L3(CH2)15(CH3)3N-AuCl4And adding the compound into 3M L0.01.01M PBS buffer solution, adding a sample containing biotin, uniformly mixing, adding into a cuvette to obtain an absorption spectrum within the wavelength range of 500-200 nm, recording the absorbance at the 267nm wavelength, and comparing the difference value of the absorbance A at the 267nm wavelength of the sample solution and the absorbance A at the 267nm wavelength of the background solution with a standard working curve to obtain the content of the biotin in the fermentation liquid.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Claims (5)
1. A method for detecting biotin, comprising: the method comprises the following steps:
a. get CH3(CH2)15(CH3)3N-AuCl4Adding the compound into PBS buffer solution, adding standard biotin to form standard biotin solutions with different concentrations, mixing uniformly, adding into a cuvette, recording the absorbance values of the standard biotin solutions with different concentrations at the wavelength of 267nm,drawing a standard working curve according to the change value of the absorbance A at the wavelength of 267nm and the concentration of biotin;
b. get CH3(CH2)15(CH3)3N-AuCl4And adding the compound into PBS buffer solution, adding the biotin sample to be detected, uniformly mixing, adding into a cuvette, recording the absorbance at the wavelength of 267nm, and calculating to obtain the concentration of the biotin sample to be detected through a standard working curve.
2. A method for detecting biotin, comprising: the method comprises the following steps:
a. get CH3(CH2)15(CH3)3N-AuCl4Adding the compound into PBS buffer solution, uniformly mixing, and adding into a cuvette to obtain an absorption spectrum with the wavelength range of 500-200 nm as a baseline;
b. get CH3(CH2)15(CH3)3N-AuCl4Adding the compound into PBS buffer solution, adding standard biotin to form standard biotin solutions with different concentrations, mixing uniformly, and adding into a cuvette to obtain absorption spectra of the biotin with different concentrations;
c. recording the absorbance values of standard biotin solutions with different concentrations at the 267nm wavelength, and drawing a standard working curve by using the change value of the absorbance A at the 267nm wavelength and the concentration of biotin;
d. get CH3(CH2)15(CH3)3N-AuCl4And adding the compound into PBS buffer solution, adding the biotin sample to be detected, uniformly mixing, adding into a cuvette, recording the absorbance at the wavelength of 267nm, and calculating to obtain the concentration of the biotin sample to be detected through a standard working curve.
3. The method for detecting biotin according to claim 1 or 2, characterized in that: the CH3(CH2)15(CH3)3N-AuCl4The composition is prepared from chloroauric acid and herba CynomoriiThe ionic surfactant cetyl trimethyl ammonium bromide is obtained by taking 10-1000 mg of cetyl trimethyl ammonium bromide, adding 50ml of water, heating, stirring and dissolving, adding a chloroauric acid solution with the concentration of 0.01-5 mg/L at room temperature, and stirring for 0.5-10 hours.
4. The method for detecting biotin according to claim 1 or 2, wherein the concentration of the standard biotin solution of different concentrations is in the range of 5 to 400. mu.g/L.
5. The method for detecting biotin according to claim 1, characterized in that: the method comprises the following steps:
a. taking CH of 0.1-2 m L3(CH2)15(CH3)3N-AuCl4Adding the compound into 3M L0.01.01M PBS buffer solution, adding standard biotin to form standard biotin solutions with different concentrations, uniformly mixing, adding into a cuvette, recording the absorbance value at 267nm, and drawing a standard working curve by using the change value of the absorbance A at 267nm and the concentration of the biotin;
b. taking CH of 0.1-2 m L3(CH2)15(CH3)3N-AuCl4And adding the compound into 3M L0.01.01M PBS buffer solution, adding the biotin sample to be detected, uniformly mixing, adding into a cuvette, recording the absorbance at the wavelength of 267nm, and calculating to obtain the concentration of the biotin sample to be detected through a standard working curve.
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| CN108801961A (en) | 2018-11-13 |
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