CN111307746A - Kit for detecting iodine concentration - Google Patents
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- CN111307746A CN111307746A CN201911300180.4A CN201911300180A CN111307746A CN 111307746 A CN111307746 A CN 111307746A CN 201911300180 A CN201911300180 A CN 201911300180A CN 111307746 A CN111307746 A CN 111307746A
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- 229910052740 iodine Inorganic materials 0.000 title claims abstract description 134
- 239000011630 iodine Substances 0.000 title claims abstract description 134
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 title claims abstract description 130
- 239000000243 solution Substances 0.000 claims abstract description 187
- 238000001514 detection method Methods 0.000 claims abstract description 161
- 239000007788 liquid Substances 0.000 claims abstract description 101
- 238000006243 chemical reaction Methods 0.000 claims abstract description 25
- -1 cerium ion Chemical class 0.000 claims abstract description 20
- 238000006479 redox reaction Methods 0.000 claims abstract description 19
- 229910052684 Cerium Inorganic materials 0.000 claims abstract description 16
- 239000003054 catalyst Substances 0.000 claims abstract description 14
- 239000000376 reactant Substances 0.000 claims abstract description 14
- OWTFKEBRIAXSMO-UHFFFAOYSA-N arsenite(3-) Chemical compound [O-][As]([O-])[O-] OWTFKEBRIAXSMO-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000011550 stock solution Substances 0.000 claims abstract description 10
- 229940006020 arsenite ion Drugs 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 65
- 238000002835 absorbance Methods 0.000 claims description 35
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 28
- 238000002156 mixing Methods 0.000 claims description 19
- KXDNMQAGVLNBKE-UHFFFAOYSA-N [AsH3].[Ce] Chemical compound [AsH3].[Ce] KXDNMQAGVLNBKE-UHFFFAOYSA-N 0.000 claims description 16
- 239000000463 material Substances 0.000 claims description 13
- 239000012488 sample solution Substances 0.000 claims description 8
- OKJMLYFJRFYBPS-UHFFFAOYSA-J tetraazanium;cerium(4+);tetrasulfate Chemical compound [NH4+].[NH4+].[NH4+].[NH4+].[Ce+4].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O OKJMLYFJRFYBPS-UHFFFAOYSA-J 0.000 claims description 8
- 150000004694 iodide salts Chemical class 0.000 abstract description 8
- 239000002699 waste material Substances 0.000 abstract description 8
- TUFLVBXBZSNFCZ-UHFFFAOYSA-N arsenic cerium Chemical compound [As].[Ce] TUFLVBXBZSNFCZ-UHFFFAOYSA-N 0.000 abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 37
- 229910001868 water Inorganic materials 0.000 description 32
- 238000000034 method Methods 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 11
- 238000001816 cooling Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- HJTAZXHBEBIQQX-UHFFFAOYSA-N 1,5-bis(chloromethyl)naphthalene Chemical compound C1=CC=C2C(CCl)=CC=CC2=C1CCl HJTAZXHBEBIQQX-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- GOLCXWYRSKYTSP-UHFFFAOYSA-N arsenic trioxide Inorganic materials O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 description 8
- GCPXMJHSNVMWNM-UHFFFAOYSA-N arsenous acid Chemical compound O[As](O)O GCPXMJHSNVMWNM-UHFFFAOYSA-N 0.000 description 8
- 238000007865 diluting Methods 0.000 description 8
- 238000004364 calculation method Methods 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 6
- XMBWDFGMSWQBCA-UHFFFAOYSA-M iodide Chemical compound [I-] XMBWDFGMSWQBCA-UHFFFAOYSA-M 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 5
- 229940006461 iodide ion Drugs 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- 239000011668 ascorbic acid Substances 0.000 description 3
- HKVFISRIUUGTIB-UHFFFAOYSA-O azanium;cerium;nitrate Chemical compound [NH4+].[Ce].[O-][N+]([O-])=O HKVFISRIUUGTIB-UHFFFAOYSA-O 0.000 description 3
- VZDYWEUILIUIDF-UHFFFAOYSA-J cerium(4+);disulfate Chemical compound [Ce+4].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O VZDYWEUILIUIDF-UHFFFAOYSA-J 0.000 description 3
- MEXSQFDSPVYJOM-UHFFFAOYSA-J cerium(4+);disulfate;tetrahydrate Chemical compound O.O.O.O.[Ce+4].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O MEXSQFDSPVYJOM-UHFFFAOYSA-J 0.000 description 3
- 229910000355 cerium(IV) sulfate Inorganic materials 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 206010067997 Iodine deficiency Diseases 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- GWXLDORMOJMVQZ-UHFFFAOYSA-N cerium Chemical compound [Ce] GWXLDORMOJMVQZ-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 2
- 235000006479 iodine deficiency Nutrition 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000036632 reaction speed Effects 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000013582 standard series solution Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- DJHGAFSJWGLOIV-UHFFFAOYSA-N Arsenic acid Chemical compound O[As](O)(O)=O DJHGAFSJWGLOIV-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000002384 drinking water standard Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052755 nonmetal Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000013139 quantization Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- PTLRDCMBXHILCL-UHFFFAOYSA-M sodium arsenite Chemical compound [Na+].[O-][As]=O PTLRDCMBXHILCL-UHFFFAOYSA-M 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000126 substance Substances 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/10—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using catalysis
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a kit for detecting iodine concentration, which comprises: the multiwell plate comprises a plurality of wells; the detection liquid A comprises arsenite ion solution; the detection solution B comprises a tetravalent cerium ion solution; the detection solution C comprises an iodine standard stock solution; when the concentration of iodine is detected, a plurality of micropores are used as a reaction container, a plurality of iodine-catalyzed arsenic-cerium redox reactions are simultaneously carried out according to preset requirements, reactants comprise arsenite ions provided by detection liquid A and tetravalent cerium ions provided by detection liquid B, and a catalyst comprises a plurality of iodides with different known concentrations provided by detection liquid C and iodides with unknown concentrations provided by sample liquid to be detected; after the reaction, detecting in a micropore detector with a preset detection wavelength, and further calculating to obtain the concentration of iodine in the sample liquid to be detected. By the mode, the iodine concentration can be simply, quickly and inexpensively detected, and waste discharge can be reduced.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to a kit for detecting iodine concentration.
Background
Water iodine is used as a human iodine nutrition evaluation index and is a main monitoring index in the prevention and treatment of iodine deficiency diseases. Taking the national life drinking water iodine content survey conducted in 2017 plus 2018 in China as an example, water iodine in more than 4 million villages and more than 12 million villages is detected in the survey, and the conservative estimation detection sample amount is nearly 40 million, so that the iodine deficiency disease prevention and treatment organization in China has great detection requirements on the water iodine.
In recent years, water iodine detection is mainly detected according to a GB/T5750.5-2006(11.1) drinking water standard detection method-inorganic nonmetal index method, the method is good in detection specificity, high in precision and accuracy, simple in instrument and equipment and suitable for conventional application in common laboratories, but arsenic trioxide used in the method is a highly toxic product. Because the current public security department strengthens strict examination and restriction on the qualification of purchasing highly toxic reagents, the purchase of arsenic trioxide (commonly called arsenic trioxide) reagents required by the arsenic-cerium catalytic luminosity standard method is difficult, and a large amount of arsenic-containing waste liquid is generated after the method is applied, thereby polluting the environment. Inductively coupled plasma mass spectrometry (ICP-MS) is high in analysis speed, and at present, a part of laboratories are used, but the instrument is expensive, the use cost is very high, and the ICP-MS is difficult to popularize and use in prefecture and county level laboratories.
Disclosure of Invention
The invention mainly solves the technical problem of providing a kit for detecting iodine concentration, which can simply, quickly and inexpensively detect the iodine concentration and reduce the waste discharge.
In order to solve the technical problems, the invention adopts a technical scheme that: providing a kit for detecting the concentration of iodine, wherein the kit is used for detecting the concentration of iodine in a sample liquid to be detected, and comprises: a porous plate comprising a plurality of micropores of the same shape, size and material, the material matching a predetermined wavelength range; the detection liquid A comprises arsenite ion solution; the detection solution B comprises a tetravalent cerium ion solution; a detection solution C, wherein the detection solution C comprises an iodine standard stock solution; when the kit is used for detecting the concentration of iodine in a sample liquid to be detected, a plurality of micropores of the porous plate are used as a reaction container for a plurality of iodine-catalyzed arsine-cerium redox reactions, and the plurality of iodine-catalyzed arsine-cerium redox reactions are simultaneously carried out according to a preset requirement, wherein the iodine-catalyzed arsine-cerium redox reactions comprise a reactant and a catalyst, the reactant comprises arsenite ions provided by the detection liquid A and tetravalent cerium ions provided by the detection liquid B, and the catalyst comprises a plurality of known iodies with different concentrations provided by the detection liquid C and iodies with unknown concentrations provided by the sample liquid to be detected; after the reaction, detecting in a micropore detector with a preset detection wavelength, and further calculating to obtain the concentration of iodine in the sample liquid to be detected, wherein the preset detection wavelength is the wavelength in the preset wavelength range, and the micropore detector is matched with the porous plate.
Wherein, the micropore detector comprises an enzyme-labeling instrument; the multi-well plate comprises a 96-well plate.
Wherein the detection liquid A comprises arsenous acid H3AsO3And (3) solution.
Wherein, the H3AsO3The concentration of the solution was 0.06 mol/L.
Wherein the detection solution B comprises a cerium ammonium sulfate solution.
Wherein the concentration of the tetravalent cerium ions is 0.025 mol/L.
Wherein the detection solution C comprises a potassium iodide solution.
Wherein the concentration of iodine is 100. mu.g/mL.
When the kit is used for detecting the concentration of iodine in a sample liquid to be detected, a first equal volume of a plurality of known different iodine concentration solutions and the sample liquid to be detected are respectively added into a plurality of micropores; then, adding the detection liquid A with the second equal volume into the micropores respectively, and fully and uniformly mixing; and then reducing the temperature of the porous plate to 0-5 ℃, sequentially adding a third equal-volume detection solution B into each micropore, uniformly mixing on a table concentrator at 25-40 ℃, and reacting until the absorbance of the micropore corresponding to the known and specified iodine concentration solution reaches a preset numerical range.
Wherein the preset detection wavelength comprises 400 nm.
The invention has the beneficial effects that: different from the situation of the prior art, the detection kit comprises a detection liquid A, a detection liquid B, a detection liquid C, a sample liquid to be detected and a multi-hole plate; when the content of iodine is detected, a plurality of micropores of a porous plate are used as a reaction container for a plurality of iodine-catalyzed arsine-cerium redox reactions, and the plurality of iodine-catalyzed arsine-cerium redox reactions are simultaneously carried out according to a preset requirement, wherein the iodine-catalyzed arsine-cerium redox reactions comprise a reactant and a catalyst, the reactant comprises arsenite ions provided by a detection liquid A and tetravalent cerium ions provided by a detection liquid B, and the catalyst comprises a plurality of iodides with different known concentrations provided by a detection liquid C and iodides with unknown concentrations provided by a sample liquid to be detected; after the reaction, detecting in a micropore detector with preset detection wavelength, and further calculating to obtain the concentration of iodine in the sample liquid to be detected, wherein the micropore detector is matched with the porous plate. Because the micropore volume is small, the required reactant amount is small, and the required catalyst amount is small, the waste discharge can be reduced, the toxicity is reduced, and the cost is also reduced; the multi-well plate (such as a 96-well plate) can simultaneously detect a plurality of samples while obtaining an iodine concentration standard curve at one time, and has small micropore volume and high reaction speed, so that the iodine concentration of the plurality of samples can be rapidly detected at one time, and the cost can be further reduced; the porous plate and the micropore detector are cheap and easy to obtain compared with an ICP-MS instrument, so that the detection cost can be reduced; the operation of reaction through the micropores of the porous plate is simple and convenient, automatic operation can be realized, and the detection speed is further improved. In addition, a set of complete micro water sample iodide detection method can be provided for samples with small sample amount, so that the detection is more standardized and unified, and the obtained experimental result is more real and reliable.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts. Wherein:
FIG. 1 is a schematic diagram showing the composition of an embodiment of the kit for detecting iodine concentration according to the present invention;
FIG. 2 is a schematic flow chart of one embodiment of the kit of FIG. 1 for detecting iodine concentration;
FIG. 3 is a schematic flow chart of another embodiment of the kit of FIG. 1 for detecting iodine concentration.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Referring to fig. 1, fig. 1 is a schematic composition diagram of a kit for detecting iodine concentration according to the present invention, the kit being used for detecting iodine concentration in a sample solution to be detected, the kit 10 comprising: the liquid sample detection apparatus includes a detection liquid A1, a detection liquid B2, a detection liquid C3, and a multi-well plate 4, and the multi-well plate 4 includes a plurality of wells 41 having the same shape, size, and material.
When the kit 10 is used for detecting the concentration of iodine in a sample solution to be detected, as shown in fig. 2, the detection is performed according to the following steps:
step S101: providing a detection liquid A, a detection liquid B, a detection liquid C, a sample liquid to be detected and a porous plate, wherein the detection liquid A comprises arsenite ion radical ion solution, the detection liquid B comprises tetravalent cerium ion solution, the detection liquid C comprises iodine standard stock solution, the porous plate comprises a plurality of micropores with the same shape, size and material, and the material is matched with a preset wavelength range.
In this embodiment, the detection liquid a includes a solution of arsenite ions, for example: arsenous acid H3AsO3Solution, sodium arsenite Na3AsO3Solutions, etc., the concentration of which is not limited, and the preparation method of which is not limited, are determined according to specific practical applications. The detection liquid B includes a solution of tetravalent cerium ions, for example: cerium ammonium sulfate (NH)4)2Ce(SO4)3Solution, ammonium cerium Nitrate (NH)4)2Ce(NO3)6Solutions, etc., the concentration of which is not limited, and the preparation method of which is not limited, are determined according to specific practical applications. The detection solution C includes an iodine standard stock solution, for example: KI standard stock solution of potassium iodide, KIO3Standard stock solutions, etc., the concentration of which is not limited, and the preparation method of which is not limited, are determined according to the specific practical application.
The multi-well plate includes a plurality of wells having the same shape, size, and material, and the wells are used as a reaction container and a cuvette (absorbance of a solution in the wells is detected at a predetermined wavelength after reaction), so the shape, size, and material of the wells are the same, and the material does not affect the measurement of the absorbance, and thus the material must be matched with a predetermined wavelength range (i.e., the material does not affect the measurement of the absorbance within the predetermined wavelength range). Commonly used multi-well plates include 16 wells, 48 wells and 96 wells, and in particular 96-well plates are more commonly used, with one being able to detect more samples at a time. The material is usually optically clear pure Polystyrene (PS). The predetermined wavelength range refers to the range of detection wavelengths, and is generally 300 nm and 700nm in the UV-visible range.
Step S102: the method comprises the steps of taking a plurality of micropores of a porous plate as a reaction container for a plurality of iodine-catalyzed arsine-cerium redox reactions, and simultaneously carrying out a plurality of iodine-catalyzed arsine-cerium redox reactions according to preset requirements, wherein the iodine-catalyzed arsine-cerium redox reactions comprise reactants and a catalyst, the reactants comprise arsenite ions provided by detection liquid A and tetravalent cerium ions provided by detection liquid B, and the catalyst comprises a plurality of iodides with different known concentrations provided by detection liquid C and iodides with unknown concentrations provided by sample liquid to be detected.
The principle of iodine catalyzing arsenic cerium redox reaction is to utilize the catalytic action of iodine on arsenic cerium redox reaction:
H3AsO3+2Ce4++H2O→H3AsO4+2Ce3++2H+
yellow Ce in the reaction4+Reduced to colorless Ce by arsenous acid3+The higher the iodine content, the faster the reaction rate, and the remaining Ce4+The less. Controlling the reaction temperature and time, and measuring the residual Ce in the reaction system at a certain wavelength4+The iodine content was determined from the absorbance of (2).
The predetermined requirements include, but are not limited to: the concentration and the amount of each reactant added in each microwell, the concentration and the amount of catalyst added (for example, how are known how are iodine with different concentrations. The present embodiment does not limit the predetermined requirements, and is determined according to the specific practical application.
Typically, the standard curve is set to not less than five known different concentrations of iodine. In one embodiment, when the detection solution C is used for preparing iodine with different concentrations, the detection solution C is diluted to the concentration of an iodine standard intermediate solution, then the highest concentration of iodine standard series of concentrations is prepared in one main micropore through the iodine standard intermediate solution, from the highest concentration solution, a low concentration solution next to the main micropore is prepared from the high concentration solution in sequence, and finally the volumes of the iodine standard series of concentrations in the main micropore are consistent. When the concentration of iodine in the sample to be detected exceeds the highest concentration range of the iodine standard series solution, the detection solution needs to be diluted, and the sample which does not exceed the highest concentration range of the iodine standard series solution directly detects the stock solution. In order to reduce errors caused by asynchronous operation, in one embodiment, arsenite ions and iodine are added firstly, then tetravalent cerium is added, the two steps need to be mixed uniformly, and the temperature needs to be reduced when the tetravalent cerium is added, and the temperature is reduced to a low temperature range of 0-5 ℃. In one application, the reaction temperature is about 30 ℃, the reaction time is 15min, or the absorbance value in the micropore corresponding to the known highest iodine concentration is about 0.15.
Step S103: after the reaction, detecting in a micropore detector with a preset detection wavelength, and further calculating to obtain the concentration of iodine in the sample liquid to be detected, wherein the preset detection wavelength is the wavelength in the preset wavelength range, and the micropore detector is matched with the porous plate.
In this embodiment, the detection wavelength is preset within a predetermined wavelength range, typically yellow Ce4+The wavelength corresponding to the maximum absorption peak of (b). For example, the predetermined detection wavelength may be 400nm, or 405nm, or 420nm, etc. In general, the predetermined detection wavelength can be determined after scanning according to the reaction system of the specific application. The multi-well plate needs to be put into a micro-well detector for detection, and therefore the multi-well plate needs to be matched with the micro-well detector so that the micro-well detector can detect the absorbance of the reaction system of each micro-well to be detected. In one application, the predetermined detection wavelength comprises 400 nm.
After the reaction, detecting in a micropore detector with preset detection wavelength, and then making a standard curve of logarithmic values of iodine concentration and absorbance value; and calculating to obtain the concentration of iodine in the sample liquid to be detected according to the standard curve and the absorbance value of the reaction system of the micropore corresponding to the sample liquid to be detected.
For example, in one embodiment, using the regression equation method in accordance with national standards, the iodine mass concentration c (μ g/L) is linear with the log of the absorbance value A:
c ═ a + blgA (or c ═ a + blnA)
Wherein c represents the mass concentration of iodine in micrograms per liter (μ g/L) in the iodine standard use series of solutions (or samples tested); a represents the intercept of a regression equation of a standard curve; b represents the slope of the regression equation of the standard curve; a represents the absorbance value determined for an iodine standard using a series of solutions (or samples tested).
And solving a regression equation of the standard curve according to the linear relation, and substituting the absorbance values of the micropores corresponding to the sample into the linear relation to solve the mass concentration of the iodine in the measured sample.
The method of the embodiment of the invention provides a detection solution A, a detection solution B, a detection solution C, a sample solution to be detected and a multi-well plate; the method comprises the following steps of taking a plurality of micropores of a porous plate as a reaction container for a plurality of iodine-catalyzed arsine-cerium redox reactions, and simultaneously carrying out a plurality of iodine-catalyzed arsine-cerium redox reactions according to preset requirements, wherein the iodine-catalyzed arsine-cerium redox reactions comprise a reactant and a catalyst, the reactant comprises arsenite ions provided by detection liquid A and tetravalent cerium ions provided by detection liquid B, and the catalyst comprises a plurality of iodides with different known concentrations provided by detection liquid C and iodides with unknown concentrations provided by sample liquid to be detected; after the reaction, detecting in a micropore detector with preset detection wavelength, and further calculating to obtain the concentration of iodine in the sample liquid to be detected, wherein the micropore detector is matched with the porous plate. Because the micropore volume is small, the required reactant amount is small, and the required catalyst amount is small, the waste discharge can be reduced, the toxicity is reduced, and the cost is also reduced; the multi-well plate (such as a 96-well plate) can simultaneously detect a plurality of samples while obtaining an iodine concentration standard curve at one time, and has small micropore volume and high reaction speed, so that the iodine concentration of the plurality of samples can be rapidly detected at one time, and the cost can be further reduced; the porous plate and the micropore detector are cheap and easy to obtain compared with an ICP-MS instrument, so that the detection cost can be reduced; the operation of reaction through the micropores of the porous plate is simple and convenient, automatic operation can be realized, and the detection speed is further improved. In addition, a set of complete micro water sample iodide detection method can be provided for samples with small sample amount, so that the detection is more standardized and unified, and the obtained experimental result is more real and reliable.
In one embodiment, the microwell detector comprises a microplate reader; the multi-well plate comprises a 96-well plate.
The embodiment adopts the microplate reader to establish the iodine detection method, has the advantages that the microplate reader is a micropore detector, and has the advantages of small sample amount, small reagent dosage, high detection speed, less generated experimental wastes and the like. Therefore, the enzyme-labeling instrument is adopted to measure the iodine, the price of the instrument and equipment is low, the use amount of the sample and the virulent reagent can be greatly reduced, the discharge of waste is reduced, and the method has important public health practical significance and practical application value for monitoring large sample amount in disease prevention and control, so that the method is easy to popularize and use in prefectural-level laboratories, and the iodine detection task is comprehensively covered. The 96-well plate is widely used, the number of the wells is large, and more samples can be detected at one time.
In one application, the detection solution A comprises arsenous acid H3AsO3And (3) solution. In particular, H3AsO3The concentration of the solution was 0.06 mol/L.
0.06mol/L of H3AsO3The method of preparing the solution may include: weighing 0.6g of arsenic trioxide, 4g of sodium chloride and 0.2g of sodium hydroxide, placing the arsenic trioxide, the sodium chloride and the sodium hydroxide into a beaker, adding about 50mL of pure water, heating the mixture until the pure water is completely dissolved, and cooling the mixture to room temperature; slowly adding 20mL of 2.5mol/L sulfuric acid solution, cooling to room temperature, diluting with pure water to 100mL, and storing in a brown bottle to obtain detection solution A, wherein the detection solution A can be stored for 6 months at room temperature.
In one application, the detection solution B includes a cerium ammonium sulfate solution. Specifically, the concentration of tetravalent cerium ions is 0.025 mol/L.
The preparation method of the 0.025mol/L cerium ammonium sulfate solution can comprise the following steps: 1.58g of ammonium ceric sulfate or 1.67g of ammonium ceric sulfate tetrahydrate were accurately weighed and dissolved in 70mL of sulfuric acid solution (c (H)2SO4) 2.5mol/L), diluted to 100mL with pure water, stored in a brown bottle and kept at room temperature for 6 months.
In one application, detection solution C comprises a potassium iodide solution. Specifically, the concentration of iodine was 100. mu.g/mL.
The preparation method of the iodine solution with 100 mug/mL can comprise the following steps: accurately weighing 0.1686g of potassium iodide which is dried to constant weight at 110 ℃ under 105 ℃, adding pure water to dissolve the potassium iodide, fixing the volume to 1000mL by using the pure water, and storing the solution in a tightly-plugged brown bottle to obtain the detection solution C, wherein the detection solution C can be stored for 6 months at 4 ℃.
Referring to fig. 3, in order to minimize operation errors and standardize the operation process, in an embodiment, in step S102, the performing a plurality of iodine-catalyzed arsine-cerium redox reactions simultaneously according to a predetermined requirement may specifically include:
step S201: first equal volumes of a plurality of solutions with known different iodine concentrations and a sample solution to be detected are respectively added into a plurality of micropores.
Step S202: then, the second equal volume of the detection liquid A is respectively added into the micropores and fully and uniformly mixed.
Step S203: then, the temperature of the multi-well plate is decreased to 0-5 ℃ (e.g., 0 ℃, 3 ℃, 5 ℃, etc.), a third equal volume of the detection solution B is sequentially added into each micro-well, and the mixture is uniformly mixed on a shaker with 25-40 ℃ (e.g., 25 ℃, 32 ℃, 40 ℃, etc.) and reacted until the absorbance of the micro-well corresponding to the known and specified iodine concentration (determined according to the specific practical application) solution reaches a preset value range.
The specification of the kit is determined according to specific application, and different specifications can be configured according to different applications. For example, for iodine monitoring applications where the number of samples to be tested is large, the specifications may be: 500 persons/cell, wherein detection liquid A (0.06mol/L H)3AsO3Solution) was 15mL 5/cartridge, assay solution B (0.025mol/L cerium ammonium sulfate solution) was 5.5mL 5/cartridge, and assay solution C (100 μ g/mL potassium iodide solution) was 10mL 1/cartridge.
Details of the kit of the invention in one application are specified below:
(1) taking out the detection solution A, the detection solution B and the detection solution C from the kit;
wherein the detection solution A is arsenous acid solution (c)(H3AsO3) 0.06 mol/L): 50mL of pure water was added to 0.6g of arsenic trioxide, 4g of sodium chloride and 0.2g of sodium hydroxide, and the mixture was dissolved by heating and then cooled to room temperature. Slowly adding 20mL of 2.5mol/L sulfuric acid solution, cooling to room temperature, diluting with pure water to 100mL, storing in brown bottle, and standing at room temperature for 6 months.
The detection solution B was a cerium ammonium sulfate solution (c (Ce)4+) 0.025 mol/L): 1.58g of ammonium ceric sulfate or 1.67g of ammonium ceric sulfate tetrahydrate were weighed and dissolved in 70mL of sulfuric acid solution (c (H)2SO4) 2.5mol/L), diluted to 100mL with pure water, stored in a brown bottle and stored at room temperature for 6 months.
Test solution C was an iodine standard stock solution (ρ (I) ═ 100 μ g/mL): 0.1686g of potassium iodide dried to constant weight at 110 ℃ under 105 ℃ is accurately weighed, dissolved by adding pure water, and the solution is fixed to 1000mL by using the pure water, stored in a brown bottle with a tight plug and can be stored in a refrigerator (4 ℃) for 6 months. When in use, 30 mu L of detection solution C is sucked and placed in a 10mL constant volume tube, and the volume is fixed to the scale by pure water, thus obtaining 300 mu g/L iodine standard intermediate solution.
(2) Sample adding:
A. selecting 8 wells as main wells in a 96-well plate, loading and diluting with 300. mu.g/L iodine standard intermediate solution and deionized water, so that the liquid in each final main well is 25. mu.L, and the concentrations of iodide ions are 300. mu.g/L, 300. mu. 2/3. mu.g/L, 300. mu. 4/9. mu.g/L, 300. mu. 8/27. mu.g/L, 300. mu. 16/81. mu.g/L, 300. mu. 32/243. mu.g/L, 300. mu. 64/729. mu.g/L, 0. mu.g/L (i.e., a plurality of known different concentrations of iodine);
B. selecting other holes in a 96-well plate as auxiliary holes, and respectively adding 25 mu L of liquid to be detected (namely a sample to be detected);
C. adding 125.0 μ L of detection solution A into the main hole and each auxiliary hole added with the solution to be detected, and mixing completely;
D. and (3) cooling the temperature of the 96-well plate to 4 ℃, accurately adding 50.0 mu L of detection liquid B into each well in sequence, starting a shaking table, and raising the temperature to 30 ℃ by a program to continue mixing and reacting.
(3) And (3) detection:
the absorbance value of each well was measured at a wavelength of 400nm when the absorbance of the first well (i.e., 300. mu.g/L in the iodine standard use series solution, i.e., a solution known to have a specified concentration of iodine) reached about 0.15.
(4) Computing
And finally, according to the iodine standard in the main hole, using the iodine concentration of the serial solutions as an abscissa, using the logarithm of the measured absorbance value A as an ordinate, drawing a standard curve in a semilogarithmic coordinate system, and checking the mass concentration of the iodine in the measured sample on the standard curve by using the absorbance value of the solution to be measured in each auxiliary hole.
Compared with the existing method for detecting iodine, the method for detecting iodine by using the kit has the following advantages:
1. compared with the traditional spectrophotometry, the kit for determining the content of iodine in a sample by using a micropore detector (enzyme-linked immunosorbent assay) greatly reduces the use amount of a detection sample and detection liquid, and avoids the use of a large amount of arsenic trioxide hypertoxic drugs and the environmental pollution caused by a large amount of toxic experimental waste liquid in the detection process.
2. The ELISA method is used for detecting samples, a 96-pore plate can be used for detecting a plurality of samples at one time, a large amount of detection time is saved, the larger the sample measuring amount is, the less the time used by a single sample is, and the support is provided for realizing the miniaturization and the kit quantization of accuracy, rapidness, small samples and high flux more easily.
3. The invention uses the enzyme-labeling instrument as a detector and uses a 96-pore plate for determination, thereby having low detection cost and high efficiency. Through the selection of detection wavelength, the determination of the linear range of the standard series, the change of the concentration and the proportion of the reagent, the optimization of the reaction time and the temperature and the like, the micro-quantification of the iodine detection sample, the rapidness of the detection time and the high-pass quantification of the detection efficiency are realized.
Advantages and features of the present invention are further described and demonstrated below in conjunction with the specific embodiments.
Wherein the detection solution A in the kit is arsenous acid solution (c (H)3AsO3) 0.06 mol/L): 50mL of pure water was added to 0.6g of arsenic trioxide, 4g of sodium chloride and 0.2g of sodium hydroxide, and the mixture was dissolved by heating and then cooled to room temperature. Slowly adding 20mL of 2.5mol/L sulfuric acid solution, cooling to room temperature, diluting with pure water to 100mL, storing in brown bottle, and standing at room temperature for 6 months.
The detection solution B was a cerium ammonium sulfate solution (c (Ce)4+) 0.025 mol/L): 1.58g of ammonium ceric sulfate or 1.67g of ammonium ceric sulfate tetrahydrate were weighed and dissolved in 70mL of sulfuric acid solution (c (H)2SO4) 2.5mol/L), diluted to 100mL with pure water, stored in a brown bottle and stored at room temperature for 6 months.
Test solution C was an iodine standard stock solution (ρ (I) ═ 100 μ g/mL): 0.1686g of potassium iodide dried to constant weight at 110 ℃ under 105 ℃ is accurately weighed, dissolved by adding pure water, and the solution is fixed to 1000mL by using the pure water, stored in a brown bottle with a tight plug and can be stored in a refrigerator (4 ℃) for 6 months.
Example 1:
in this example, a sample with a known concentration of 50.9. + -. 2.3. mu.g/L was used as the first sample to be tested (i.e., the sample to be tested), 8 main wells were used as the standard curve test series, and 24 auxiliary wells were used to test the first sample to be tested.
(1) Preparation of standard solution:
and taking out the detection solution A, the detection solution B and the detection solution C in the kit for later use.
When in use, 30 mu L of detection solution C is sucked and placed in a 10mL constant volume tube, and the volume is fixed to the scale by pure water, thus obtaining 300 mu g/L iodine standard intermediate solution.
Taking the solution with the environmental standard sample batch number B1903109, accurately sucking 0.4mL, diluting to a constant volume of 10mL to obtain a liquid with the concentration of 5.09 +/-0.23 mg/L, taking 100.0 mu L of the liquid, placing the liquid in a constant volume tube of 10mL, diluting to a constant volume with pure water to a scale, and obtaining a first solution to be detected with the concentration of 50.9 +/-2.3 mu g/L.
(2) Sample adding:
A. selecting 8 wells from a 96-well plate as main wells, adding 75 mu L of iodine standard intermediate solution with the concentration of 300 mu g/L into the 1 st well, adding 25 mu L of deionized water into the subsequent 2 nd to 7 th wells, accurately sucking 50 mu L of iodine standard intermediate solution into the 1 st well by using a microsyringe, adding 50 mu L of iodine standard intermediate solution into the 2 nd well, sufficiently mixing the solution, accurately sucking 50 mu L of iodine standard intermediate solution from the 2 nd well, adding the solution into the 3 rd well, sufficiently mixing the solution, repeating the steps, sucking 50 mu L of liquid in the seventh well, discarding the liquid, so that the liquid in the final main wells is 25 mu L, and the concentrations of iodine ions are respectively 300 mu g/L, 300 mu 2/3 mu g/L, 300 mu 4/9 mu g/L, 300 mu 8/27 mu g/L, 300 mu 16/81 mu g/L, 300 mu g 32/243 mu g/L, 300 mu g/L, 64/729 mu g/L, 0 mu g/L;
B. respectively adding 25 mu L of the first solution to be detected into the other rows of auxiliary holes selected from the 96-well plate;
C. respectively adding 125.0 μ L of detection solution A into the main hole and each auxiliary hole added with the first solution to be detected, and fully and uniformly mixing;
D. and (3) cooling the temperature of the 96-well plate to 4 ℃, accurately adding 50.0 mu L of detection liquid B into each well in sequence, starting a shaking table, programming the temperature to 30 ℃, and continuing to uniformly mix and react for about 25 min.
(3) And (3) detection:
when the absorbance of the first well (i.e., 300. mu.g/L in the iodine standard use series solution, i.e., a known and specified iodine concentration solution) reached about 0.15, the absorbance value of each well was measured at a wavelength of 400nm, and the results of the measurements are shown in Table 1.
TABLE 1 EXAMPLE 1 detection of absorbance value (400nm) of solution I to be detected
(4) Computing
According to the known concentration values of iodine content in the wells from the 1 st columns A to H of 300. mu.g/L, 300. mu. 2/3. mu.g/L, 300. mu. 4/9. mu.g/L, 300. mu. 8/27. mu.g/L, 300. mu. 16/81. mu.g/L, 300. mu. 32/243. mu.g/L, 300. mu. 64/729. mu.g/L and 0. mu.g/L as abscissa, the logarithm of the measured absorbance value A is plotted as ordinate, and a standard curve having the regression equation of y-0.0042 x +0.3936 and the correlation coefficient R is plotted in a semilogarithmic coordinate system2＝0.9991。
The concentration values of the iodide ion content of the 24 samples to be detected are calculated according to the regression equation, and the calculation results are shown in the following table 2.
TABLE 2 EXAMPLE 1 detection of iodine ion concentration value (. mu.g/L) of solution I to be detected
| Number of sample application hole | First row (Main hole row) | Second row (auxiliary hole row) | Third row (auxiliary hole row) | Fourth row (auxiliary hole row) |
| A | 300 | 49.1 | 50.5 | 49.9 |
| B | 300*2/3 | 50.1 | 51.2 | 52.1 |
| C | 300*4/9 | 52.4 | 48.9 | 53.0 |
| D | 300*8/27 | 50.6 | 49.0 | 53.0 |
| E | 300*16/81 | 48.8 | 51.0 | 50.3 |
| F | 300*32/243 | 49.9 | 49.9 | 53.3 |
| G | 300*64/729 | 53.0 | 48.3 | 48.8 |
| H | 0 | 52.2 | 51.9 | 53.5 |
The average iodine content concentration of the first solution to be tested is 50.9 mug/L, the standard deviation is 1.6 mug/L, and the Coefficient of Variation (CV) is 3.1 percent according to the calculation result.
Example 2:
in this example, a sample with a known concentration of 50.9. + -. 2.3. mu.g/L was used as the second sample, 8 main wells were used as the standard curve test array, and 24 auxiliary wells were used to detect the second sample.
(1) Preparation of standard solution:
and taking out the detection solution A, the detection solution B and the detection solution C in the kit for later use.
When in use, 30 mu L of detection solution C is sucked and placed in a 10mL constant volume tube, and the volume is fixed to the scale by pure water, thus obtaining 300 mu g/L iodine standard intermediate solution.
And (3) taking a liquid with the concentration of 5.09 +/-0.23 mg/L obtained by diluting the environment standard sample, putting 100.0 mu L of the liquid into a 10mL constant volume tube, and fixing the volume to a scale by using pure water to obtain a liquid II to be detected with the concentration of 50.9 +/-2.3 mu g/L.
(2) Sample adding:
A. selecting 8 wells from a 96-well plate as main wells, adding 75 mu L of iodine standard intermediate solution with the concentration of 300 mu g/L into the 1 st well, adding 25 mu L of deionized water into the subsequent 2 nd to 7 th wells, accurately sucking 50 mu L of iodine standard intermediate solution into the 1 st well by using a microsyringe, adding 50 mu L of iodine standard intermediate solution into the 2 nd well, sufficiently mixing the solution, accurately sucking 50 mu L of iodine standard intermediate solution from the 2 nd well, adding the solution into the 3 rd well, sufficiently mixing the solution, repeating the steps, sucking 50 mu L of liquid in the seventh well, discarding the liquid, so that the liquid in the final main wells is 25 mu L, and the concentrations of iodine ions are respectively 300 mu g/L, 300 mu 2/3 mu g/L, 300 mu 4/9 mu g/L, 300 mu 8/27 mu g/L, 300 mu 16/81 mu g/L, 300 mu g 32/243 mu g/L, 300 mu g/L, 64/729 mu g/L, 0 mu g/L;
B. respectively adding 25 mu L of a second liquid to be detected into the other rows of auxiliary holes selected from the 96-well plate;
C. adding 125.0 μ L of detection solution A into the main hole and each auxiliary hole added with the second solution to be detected, and mixing completely;
D. and (3) cooling the temperature of the 96-well plate to 4 ℃, accurately adding 50.0 mu L of detection liquid B into each well in sequence, starting a shaking table, programming the temperature to 30 ℃, and continuing to uniformly mix and react for about 25 min.
(3) And (3) detection:
when the absorbance of the first well (i.e., 300. mu.g/L in the iodine standard use series solution) reached about 0.15, the absorbance value of each well was measured at a wavelength of 400nm, and the results are shown in Table 3.
TABLE 3 example 2 Absorbance value (400nm) of test sample II
| Number of sample application hole | First row (Main hole row) | Second row (auxiliary hole row) | Third row (auxiliary hole row) | Fourth row (auxiliary hole row) |
| A | 0.13 | 1.506 | 1.485 | 1.506 |
| B | 0.339 | 1.467 | 1.515 | 1.462 |
| C | 0.67 | 1.49 | 1.468 | 1.496 |
| D | 0.987 | 1.469 | 1.526 | 1.498 |
| E | 1.35 | 1.509 | 1.499 | 1.511 |
| F | 1.717 | 1.466 | 1.495 | 1.534 |
| G | 1.951 | 1.519 | 1.509 | 1.478 |
| H | 2.446 | 1.492 | 1.495 | 1.487 |
(4) And (3) calculating:
according to the known concentration values of iodine content in the wells from the 1 st columns A to H of 300. mu.g/L, 300. mu. 2/3. mu.g/L, 300. mu. 4/9. mu.g/L, 300. mu. 8/27. mu.g/L, 300. mu. 16/81. mu.g/L, 300. mu. 32/243. mu.g/L, 300. mu. 64/729. mu.g/L and 0. mu.g/L as abscissa, the logarithm of the measured absorbance value A is plotted as ordinate, and a standard curve having the regression equation of y-0.0043 x +0.3917 and the correlation coefficient R is plotted in a semilogarithmic coordinate system2＝0.9994。
And (3) calculating the iodine content concentration values of the 24 samples II to be detected according to the regression equation, wherein the calculation result is shown in the following table 4.
TABLE 4 example 2 detection of iodine concentration value (. mu.g/L) of solution II to be detected
| Number of sample application hole | First row (Main hole row) | Second row (auxiliary hole row) | Third row (auxiliary hole row) | Fourth row (auxiliary hole row) |
| A | 300 | 49.7 | 51.2 | 49.7 |
| B | 300*2/3 | 52.4 | 49.1 | 52.7 |
| C | 300*4/9 | 50.8 | 52.3 | 50.4 |
| D | 300*8/27 | 52.3 | 48.4 | 50.3 |
| E | 300*16/81 | 49.5 | 50.2 | 49.4 |
| F | 300*32/243 | 52.5 | 50.5 | 47.9 |
| G | 300*64/729 | 48.9 | 49.5 | 51.6 |
| H | 0 | 50.7 | 50.5 | 51.0 |
The average iodine content concentration of the solution II to be detected is 50.5 mug/L, the standard deviation is 1.3 mug/L, and the Coefficient of Variation (CV) is 2.6 percent according to the calculation result.
Example 3:
in this example, a sample with a known concentration of 124. + -. 12.4. mu.g/L was used as the third analyte, 8 main wells were used as the standard curve test array, and 24 auxiliary wells were used to detect the third analyte.
(1) Preparation of standard solution:
and taking out the detection solution A, the detection solution B and the detection solution C in the kit for later use.
When in use, 30 mu L of detection solution C is sucked and placed in a 10mL constant volume tube, and the volume is fixed to the scale by pure water, thus obtaining 300 mu g/L iodine standard intermediate solution.
Taking solution of the environmental standard sample, accurately sucking 0.2mL, and metering to a 10mL constant volume tube to obtain a solution III to be detected with a concentration of 124 +/-12.4 g mu g/L.
(2) Sample adding:
A. selecting 8 wells from a 96-well plate as main wells, adding 75 mu L of iodine standard intermediate solution with the concentration of 300 mu g/L into the 1 st well, adding 25 mu L of deionized water into the subsequent 2 nd to 7 th wells, accurately sucking 50 mu L of iodine standard intermediate solution into the 1 st well by using a microsyringe, adding 50 mu L of iodine standard intermediate solution into the 2 nd well, sufficiently mixing the solution, accurately sucking 50 mu L of iodine standard intermediate solution from the 2 nd well, adding the solution into the 3 rd well, sufficiently mixing the solution, repeating the steps, sucking 50 mu L of liquid in the seventh well, discarding the liquid, so that the liquid in the final main wells is 25 mu L, and the concentrations of iodine ions are respectively 300 mu g/L, 300 mu 2/3 mu g/L, 300 mu 4/9 mu g/L, 300 mu 8/27 mu g/L, 300 mu 16/81 mu g/L, 300 mu g 32/243 mu g/L, 300 mu g/L, 64/729 mu g/L, 0 mu g/L;
B. respectively adding 25 mu L of liquid III to be detected into the other rows of auxiliary holes selected from the 96-well plate;
C. adding 125.0 μ L of detection solution A into the main hole and each auxiliary hole added with the solution III to be detected, and mixing completely;
D. and (3) cooling the temperature of the 96-well plate to 4 ℃, accurately adding 50.0 mu L of detection liquid B into each well in sequence, starting a shaking table, programming the temperature to 30 ℃, and continuing to uniformly mix and react for about 25 min.
(3) And (3) detection:
when the absorbance of the first well (i.e., 300. mu.g/L in the iodine standard use series solution) reached about 0.15, the absorbance value of each well was measured at a wavelength of 400nm, and the results are shown in Table 5.
TABLE 5 EXAMPLE 3 Absorbance value (400nm) of sample solution III
| Number of sample application hole | First row (Main hole row) | Second row (auxiliary hole row) | Third row (auxiliary hole row) | Fourth step ofColumn (auxiliary hole column) |
| A | 0.15 | 0.773 | 0.753 | 0.726 |
| B | 0.369 | 0.816 | 0.742 | 0.744 |
| C | 0.671 | 0.827 | 0.851 | 0.717 |
| D | 0.978 | 0.833 | 0.828 | 0.767 |
| E | 1.352 | 0.754 | 0.754 | 0.817 |
| F | 1.617 | 0.737 | 0.762 | 0.697 |
| G | 1.851 | 0.846 | 0.772 | 0.774 |
| H | 2.346 | 0.797 | 0.816 | 0.725 |
(4) Computing
According to the known concentration values of iodine content in the wells A to H in the columns 1, 300. mu.g/L, 300. mu. 2/3. mu.g/L, 300. mu. 4/9. mu.g/L, 300. mu. 8/27. mu.g/L, 300. mu. 16/81. mu.g/L, 300. mu. 32/243. mu.g/L, 300. mu. 64/729. mu.g/L and 0. mu.g/L, the logarithm of the measured absorbance value A is plotted in a semilogarithmic coordinate system, the regression equation of the standard curve is-0.004 x +0.3642, and the correlation coefficient R is2＝0.9995。
And (3) respectively calculating the concentration values of the iodide ion content of the 24 samples to be detected according to the regression equation, wherein the calculation results are shown in the following table 6.
TABLE 6 example 3 detection of iodide ion concentration value (. mu.g/L) of solution III to be detected
The average iodine content concentration of the solution III to be detected is 118.7 μ g/L, the standard deviation is 6.2 μ g/L, and the Coefficient of Variation (CV) is 5.2%
Example 4:
in this example, a sample with a known concentration of 50.9. + -. 2.3. mu.g/L was used as the fourth test solution, 8 main wells were used as the standard curve test column, 24 auxiliary wells were used for the fourth test solution, and a 50. mu.g/L solution was added.
(1) Preparation of standard solution:
and taking out the detection solution A, the detection solution B and the detection solution C in the kit for later use.
When in use, 30 mu L of detection solution C is sucked and placed in a 10mL constant volume tube, and the volume is fixed to the scale by pure water, thus obtaining 300 mu g/L iodine standard intermediate solution.
Taking a liquid with the concentration of 5.09 +/-0.23 mg/L obtained by diluting an environmental standard sample, taking 100.0 mu L of the liquid, placing the liquid in a 10mL constant volume tube, and using pure water to fix the volume to a scale to obtain a to-be-detected liquid IV with the concentration of 50.9 +/-2.3 mu g/L.
1mL of 300. mu.g/L iodine standard intermediate solution was taken, and 5mL of water was added to obtain a standard solution having a concentration of 50. mu.g/L.
(2) Sample adding:
A. selecting 8 wells from a 96-well plate as main wells, adding 75 mu L of iodine standard intermediate solution with the concentration of 300 mu g/L into the 1 st well, adding 25 mu L of deionized water into the subsequent 2 nd to 7 th wells, accurately sucking 50 mu L of iodine standard intermediate solution into the 1 st well by using a microsyringe, adding 50 mu L of iodine standard intermediate solution into the 2 nd well, sufficiently mixing the solution, accurately sucking 50 mu L of iodine standard intermediate solution from the 2 nd well, adding the solution into the 3 rd well, sufficiently mixing the solution, repeating the steps, sucking 50 mu L of liquid in the seventh well, discarding the liquid, so that the liquid in the final main wells is 25 mu L, and the concentrations of iodine ions are respectively 300 mu g/L, 300 mu 2/3 mu g/L, 300 mu 4/9 mu g/L, 300 mu 8/27 mu g/L, 300 mu 16/81 mu g/L, 300 mu g 32/243 mu g/L, 300 mu g/L, 64/729 mu g/L, 0 mu g/L;
B. and respectively adding 25 mu L of liquid to be tested into a second row of attachment holes in a 96-well plate. Adding 25 mul of the liquid to be detected and 25 mul of the standard solution with the concentration of 50 mug/L into the third row of auxiliary holes respectively, taking out 25 mul of the liquid to be detected after uniformly mixing, and placing the liquid in the fourth row of auxiliary holes;
C. adding 125.0 μ L of detection solution A into the main hole and each auxiliary hole added with the solution to be detected, and mixing completely;
D. and (3) cooling the temperature of the 96-well plate to 4 ℃, accurately adding 50.0 mu L of detection liquid B into each well in sequence, starting a shaking table, programming the temperature to 30 ℃, and continuing to uniformly mix and react for about 25 min.
(3) And (3) detection:
when the absorbance of the first well (i.e., 300. mu.g/L in the iodine standard use series solution) reached about 0.15, the absorbance value of each well was measured at a wavelength of 400nm, and the results are shown in Table 7.
TABLE 7 EXAMPLE 4 Absorbance value (400nm) of sample solution III
| Number of sample application hole | First row (Main hole row) | Second row (auxiliary hole row) | Third row (auxiliary hole row) | Fourth row (auxiliary hole row) |
| A | 0.159 | 1.497 | 1.523 | 1.486 |
| B | 0.379 | 1.518 | 1.512 | 1.491 |
| C | 0.701 | 1.519 | 1.495 | 1.504 |
| D | 1.062 | 1.502 | 1.476 | 1.534 |
| E | 1.392 | 1.527 | 1.519 | 1.476 |
| F | 1.654 | 1.489 | 1.468 | 1.529 |
| G | 1.925 | 1.484 | 1.497 | 1.509 |
| H | 2.395 | 1.525 | 1.489 | 1.517 |
(4) And (3) calculating:
according to the known concentration values of iodine content in the wells A to H of the columns 1, 300. mu.g/L, 300. mu. 2/3. mu.g/L, 300. mu. 4/9. mu.g/L, 300. mu. 8/27. mu.g/L, 300. mu. 16/81. mu.g/L, 300. mu. 32/243. mu.g/L, 300. mu. 64/729. mu.g/L, 0. mu.g/L, the logarithm of the measured absorbance value A is the ordinate, and a standard curve is plotted in a semilogarithmic coordinate system, the standard curve being obtained by plotting the logarithm of the measured absorbance value A in the semilogarithmic coordinate systemThe regression equation for the standard curve is-0.004 x +0.3783 with a correlation coefficient R2＝0.9997。
And (3) respectively calculating the concentration values of the iodide ion content of the 24 solutions to be detected according to the regression equation, wherein the calculation results are shown in the following table 8.
TABLE 8 example 4 detection of iodine ion concentration value (. mu.g/L) of solution four to be tested
| Number of sample application hole | First row (Main hole row) | Second row (auxiliary hole row) | Third row (auxiliary hole row) | Fourth row (auxiliary hole row) |
| A | 300 | 50.8 | 48.9 | 51.6 |
| B | 300*2/3 | 52.2 | 49.7 | 51.2 |
| C | 300*4/9 | 49.2 | 50.9 | 50.3 |
| D | 300*8/27 | 50.4 | 52.3 | 48.1 |
| E | 300*16/81 | 49.3 | 49.2 | 52.3 |
| F | 300*32/243 | 49.9 | 52.9 | 48.5 |
| G | 300*64/729 | 51.7 | 50.8 | 49.9 |
| H | 0 | 48.8 | 51.4 | 49.3 |
The average iodine content concentration of the solution four to be detected is 50.3 mug/L, the standard deviation is 1.2 mug/L, and the Coefficient of Variation (CV) is 2.4 percent according to the calculation result; after the standard addition, the average iodine content concentration is 50.4 mug/L, the standard deviation is 1.4 mug/L, and the Coefficient of Variation (CV) is 2.8%. The calculated recovery was (50.4 × 2-50.3)/50 ═ 101.0%.
As can be seen from the above examples, the kit of the present invention has good accuracy, reproducibility, etc. in detecting iodine content.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A kit for detecting iodine concentration is characterized in that the kit is used for detecting the concentration of iodine in a sample liquid to be detected, and comprises:
a porous plate comprising a plurality of micropores of the same shape, size and material, the material matching a predetermined wavelength range;
the detection liquid A comprises arsenite ion solution;
the detection solution B comprises a tetravalent cerium ion solution;
a detection solution C, wherein the detection solution C comprises an iodine standard stock solution;
when the kit is used for detecting the concentration of iodine in a sample liquid to be detected, a plurality of micropores of the porous plate are used as a reaction container for a plurality of iodine-catalyzed arsine-cerium redox reactions, and the plurality of iodine-catalyzed arsine-cerium redox reactions are simultaneously carried out according to a preset requirement, wherein the iodine-catalyzed arsine-cerium redox reactions comprise a reactant and a catalyst, the reactant comprises arsenite ions provided by the detection liquid A and tetravalent cerium ions provided by the detection liquid B, and the catalyst comprises a plurality of known iodies with different concentrations provided by the detection liquid C and iodies with unknown concentrations provided by the sample liquid to be detected; after the reaction, detecting in a micropore detector with a preset detection wavelength, and further calculating to obtain the concentration of iodine in the sample liquid to be detected, wherein the preset detection wavelength is the wavelength in the preset wavelength range, and the micropore detector is matched with the porous plate.
2. The kit of claim 1, wherein the microwell detector comprises a microplate reader; the multi-well plate comprises a 96-well plate.
3. The kit according to claim 1, wherein the detection solution A comprises arsenite H3AsO3And (3) solution.
4. The kit of claim 3, wherein H is3AsO3The concentration of the solution was 0.06 mol/L.
5. The kit according to claim 1, wherein the detection solution B comprises a cerium ammonium sulfate solution.
6. The kit according to claim 5, wherein the concentration of the tetravalent cerium ion is 0.025 mol/L.
7. The kit according to claim 1, wherein the detection solution C comprises a potassium iodide solution.
8. The kit of claim 7, wherein the concentration of iodine is 100 μ g/mL.
9. The kit according to claim 1, wherein when the kit is used for detecting the concentration of iodine in a sample solution to be detected, a first equal volume of a plurality of solutions with known different iodine concentrations and the sample solution to be detected are added into a plurality of micropores respectively; then, adding the detection liquid A with the second equal volume into the micropores respectively, and fully and uniformly mixing; and then reducing the temperature of the porous plate to 0-5 ℃, sequentially adding a third equal-volume detection solution B into each micropore, uniformly mixing on a table concentrator at 25-40 ℃, and reacting until the absorbance of the micropore corresponding to the known and specified iodine concentration solution reaches a preset numerical range.
10. The kit of claim 1, wherein the predetermined detection wavelength comprises 400 nm.
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Application publication date: 20200619 |