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WO2013189265A1 - Procédé, système et kit de détection de la concentration en ion potassium - Google Patents

Procédé, système et kit de détection de la concentration en ion potassium Download PDF

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Publication number
WO2013189265A1
WO2013189265A1 PCT/CN2013/077335 CN2013077335W WO2013189265A1 WO 2013189265 A1 WO2013189265 A1 WO 2013189265A1 CN 2013077335 W CN2013077335 W CN 2013077335W WO 2013189265 A1 WO2013189265 A1 WO 2013189265A1
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Prior art keywords
wavelength
solution
sample
concentration
buffer
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PCT/CN2013/077335
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English (en)
Chinese (zh)
Inventor
唐亚林
孙红霞
杨千帆
向俊锋
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中国科学院化学研究所
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Priority claimed from CN201210207722.5A external-priority patent/CN102735623B/zh
Priority claimed from CN201210206227.2A external-priority patent/CN102735664B/zh
Priority claimed from CN201210205861.4A external-priority patent/CN102866149B/zh
Priority claimed from CN201210205843.6A external-priority patent/CN102866148B/zh
Application filed by 中国科学院化学研究所 filed Critical 中国科学院化学研究所
Publication of WO2013189265A1 publication Critical patent/WO2013189265A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • Ion-selective electrode method Determination of potassium and sodium ions in serum and urine on a dedicated instrument. Because it has the advantages of low sample consumption, fast and accurate, and easy operation, it is the most convenient and accurate method among all the current methods, and there is almost a tendency to replace other methods.
  • the principle is:
  • the ion selective electrode is an electrochemical sensor with a sensitive membrane electrode that selectively responds to specific ions, converting the ion activity into a potential signal, within a certain range, its potential and solution.
  • the logarithm of the specific ion activity is linear.
  • the ion activity of the unknown solution can be obtained by comparison with the solution of the known ion concentration. According to the measurement process, it is divided into the direct measurement method and the indirect measurement method. The measurement method is performed by diluting the sample to be tested by an indirect measurement method, and the measured ion activity is closer to the ion concentration.
  • Enzymatic method The principle of enzymatic determination of potassium is to use the activation of pyruvate kinase, which catalyzes the conversion of phosphoenolpyruvate to lactic acid accompanied by the consumption of reduced coenzyme I, and measures NADH at a wavelength of 340 nm. The absorbance drops.
  • the excitation intensity of the wavelength at the third wavelength is detected using an excitation wavelength of 560 nm, wherein the first wavelength is in the range of 560 nm to 590 nm, and the second wavelength is in the range of 500 nm to 540 nm, and the third wavelength is 580nm to 640nm range;
  • step (4) placing the test solution obtained in the step (4) under an ultraviolet visible light absorption spectrometer or a spectrophotometer, Detecting the absorbance value of the test solution at the first wavelength and the second wavelength, or placing the test solution under a fluorescence spectrometer, and detecting the fluorescence intensity value at the third wavelength by using an excitation wavelength of 560 nm;
  • a second aspect of the invention provides a method of detecting potassium ion concentration in a liquid sample in the context of sodium ions, the method comprising the steps of:
  • each of the solution samples contains the same concentration of a DNA molecule capable of forming a G-quadruplex, the same concentration of sodium Ions and the same concentration of cyanine dyes;
  • the fluorescence intensity value of the wavelength at the third wavelength is detected by a fluorescence spectrometer using an excitation wavelength of 560 nm, wherein the first wavelength is in the range of 560 nm to 590 nm, and the fourth wavelength is in the range of 610 nm to 670 nm, the third wavelength In the range of 580 nm to 640 nm;
  • a third aspect of the invention provides a method of detecting a range of potassium ion concentrations in a liquid sample, the method comprising the steps of:
  • the buffer is selected from the group consisting of tris buffer, hydrochloric acid-borax buffer, triethanolamine buffer, imidazole-hydrochloric acid buffer, and glycine buffer Or 2-amino-2-methyl- ⁇ propanol buffer.
  • the buffer is selected from the group consisting of trishydroxymethylaminomethane
  • the concentration of the buffer in the buffer is not particularly limited, but a preferred concentration range is 10 to 50 mmol/L.
  • R 6 is alkyl, phenyl, alkyl-substituted phenyl
  • R 2, R 3, and R 5 are independently selected from H or dC 6 alkyl, or R 2 and R 3 are attached to them
  • the carbon atoms together form a 5- to 7-membered ring structure, or form a 5- to 7-membered ring structure together with R 5 and the carbon atom to which they are attached
  • R 7 is a dC 6 alkyl or sulfonate substituted dC 6 alkyl
  • Y is a counter ion, and varies according to the charge of R 7 .
  • R 7 is an alkyl group
  • Y is a halogen anion; if only one of R 7 has a sulfonate, Y is not required.
  • a counterion if both R 7 and a sulfonate, Y is a triethylamine cation;
  • Xi, X 2 is independently selected from carbon (C), oxygen (0), sulfur (S), selenium (Se) or ⁇ (Te).
  • the method according to the first, second and third aspects of the invention which is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, iso Butyl, n-hexyl, isohexyl, phenyl, methylphenyl or dimethylphenyl.
  • R 2 , R 3 , and R 5 are independently selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, and iso Butyl, tert-butyl, pentyl, isopentyl, n-hexyl or isohexyl.
  • the plurality of solution samples can be formulated by using a soluble potassium salt such as potassium chloride, potassium sulfate, potassium nitrate or the like, potassium ions in each solution sample Concentration
  • a soluble potassium salt such as potassium chloride, potassium sulfate, potassium nitrate or the like
  • potassium ions in each solution sample Concentration The range is preferably in the range of 0 to 300 mmol/L, further preferably in the range of 0 to 200 mmol/L, still more preferably in the range of 0 to 150 mmol/L, and most preferably in the range of 20 to 100 mmol/L, wherein
  • Non-limiting examples of soluble potassium salts include potassium chloride, potassium bromide, potassium iodide, potassium sulfate or potassium nitrate.
  • the concentration of sodium ions may be adjusted by adding a soluble sodium salt or using a buffer containing sodium ions, non-limiting examples of which include , sodium chloride, sodium bromide, sodium iodide, sodium sulfate or sodium nitrate.
  • the concentration of the cyanine dye in the solution sample is in the range of 3 to 20 ⁇ m ⁇ 1 / ⁇ , preferably 5 to ⁇ / ⁇ , which is capable of forming G
  • concentration of the tetra-stranded DNA molecule in the solution sample is in the range of 3 to 30 ⁇ m ⁇ 1 / ⁇ , preferably 5 to 20 ⁇ 1 / ⁇ , and further preferably 10 to 20 ⁇ 1 / ⁇ .
  • TTAGGGTTAGGG TTAGGGTTAGGG, TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG, AGGGTTAGGGTTAGGGTTAGGG, TGAGGGTGGGGAGGGTGGGGAA, AGGGAGGGCGCTGGGAGGAGGG, GGGCGCGGGAGGAATTGGGCGGG, GGTTGGTGTGGTTGG, TTGGGGTTGGGGTTGGGG, TTGGGGTTGGGG, GGGGTTGGGG, GGGCGCGGGAGGAAGGGGGCGGG or
  • a DNA molecule such as GGGCGCGGGAGGAATTGGGCGGG, but the range of DNA sequence ⁇ ij which can form a G-quadruplex is not limited by these enumerations.
  • the length of the DNA molecule capable of forming a G-quadruplex used in the present invention is not particularly limited, but is preferably 6 300 bases in length, more preferably 10 100 bases in length, and most preferably 10 30 The length of each base.
  • a fourth aspect of the present invention provides a kit for carrying out the method of the present invention, the kit comprising: a buffer having a pH of 6.2 to 8.2, a soluble potassium salt, a DNA molecule capable of forming a G-quadruplex, and a cyanine dye.
  • the kit according to the fourth aspect of the present invention further comprising a standard color chart, wherein different colors in the standard color chart correspond to different potassium ion concentrations.
  • the color on the standard color chart can be determined by: (i) preparing a plurality of solution samples having different potassium ion concentrations using a buffer solution having a pH of 6.2 to 8.2, so that each of the solution samples contains the same concentration of a DNA molecule capable of forming a G quadruplex, and the same concentration of sodium ions.
  • the preparation method can be referred to the synthetic route described in Leslie GS, Brooker and Frank LW, JACS, 1935, 547-551, or may be used. Other methods well known in the art are prepared.
  • a fifth aspect of the invention provides a system for carrying out the method of the first and second aspects of the invention, the system comprising the kit of the fourth aspect of the invention and an ultraviolet visible light absorption spectrometer or a spectrophotometer or a fluorescence spectrometer.
  • the present invention uses a cyanine dye supramolecular probe, which is sensitive to the change of potassium-regulated G-quadruplex structure, accompanied by a change in aggregate morphology, and exhibits an absorption band up to a displacement of nearly one hundred nanometers in the ultraviolet absorption spectrum. , thus producing a change in color, enabling visual detection;
  • the cyanine dye supramolecules used in the present invention can produce significant changes in both ultraviolet absorption spectrum and fluorescence spectrum, and can be quantitatively detected by an ordinary ultraviolet absorption spectrometer or a spectrophotometer or a fluorescence spectrometer, and no special or additional instruments are required. , the test cost is low, and it is convenient for promotion and application in the industry;
  • the reagent components used in the present invention are only 3 to 4 kinds, and can be detected by instruments only when mixed in proportion, and the operation is simple, quick, and low in cost, and the system operates in a buffer environment without polluting the environment.
  • the reagents used in the invention are simple in composition, small in variety, have no influence on each other, and have good stability, and can be stored for a long time, which can ensure the application test effect well;
  • FIG. 1 is a potassium ion concentration standard curve according to Example 1 of the present invention.
  • Figure 2 is a standard curve of potassium ion concentration according to Example 2 of the present invention.
  • Figure 3 is a standard curve of potassium ion concentration according to Example 3 of the present invention.
  • Figure 4 is a standard curve of potassium ion concentration according to Example 4 of the present invention.
  • Figure 5 is a potassium ion concentration standard curve according to Example 9 of the present invention.
  • FIG 8 is a standard curve of potassium ion concentration according to Example 12 of the present invention.
  • the present invention will be described in more detail by way of specific embodiments thereof with reference to the accompanying drawings. The present invention may be practiced by those skilled in the art, and the scope of the invention should not be limited to the specific embodiments set forth herein.
  • the instruments used in the examples of the present invention are: UV-visible absorption spectrometer, model Agilent 8453 UV-visible spectrophotometer; fluorescence spectrometer, model Hitachi F4500 spectrofluorometer (Japan).
  • the DNA capable of forming a G-quadruplex used in the present example is AGGGTTAGGGTTAGGGTTAGGG, and the cyanine dye used is a compound of the following formula
  • a certain amount of DNA was dissolved in Tris-HCl buffer at pH 6.2 to prepare a mother liquor at a concentration of 200 ⁇ / ⁇ DNA for use.
  • the above samples were each analyzed by an ultraviolet absorption spectrometer. Everything is done at room temperature without additional conditions.
  • the ultraviolet absorption spectrum collects data at a wavelength of 400 to 800 nm.
  • the absorbance (at the 580 nm) of the standard sample is plotted on the abscissa and the potassium ion concentration of the standard sample is plotted on the ordinate to obtain a standard curve of the potassium ion concentration, as shown in Fig. 1.
  • the potassium ion concentration of the corresponding test solution can be found on the standard curve, and this value is divided by 1% to obtain the potassium ion concentration value of the urine sample to be tested.
  • Table 1 The results are shown in Table 1 below.
  • the DNA which can be used to form the G quadruplex in this example is TGAGGGTGGGG
  • a certain amount of DNA was dissolved in a boric acid-borax buffer solution having a pH of 8.2 to prepare a mother liquor having a concentration of 200 ⁇ m ⁇ 1 / ⁇ DNA, and was used.
  • the above samples were each analyzed by an ultraviolet absorption spectrometer. Everything is done at room temperature without additional conditions.
  • the ultraviolet absorption spectrum collects data at a wavelength of 400 to 800 nm.
  • the DNA which can be used to form the G four chain in this example is GGGCCAGGGAG compound.
  • the above samples were each analyzed by an ultraviolet absorption spectrometer. Everything is done at room temperature without additional conditions.
  • the ultraviolet absorption spectrum collects data at a wavelength of 400 to 800 nm.
  • the DNA capable of forming a G quadruplex used in the present example is AGGGTT, and the cyanine dye used is a compound of the following formula
  • a certain amount of DNA was dissolved in a pH 7.0 7.0 Tris-HCl buffer to prepare a 1.2 mmol/L DNA stock solution for use.
  • the fluorescence intensity (FI) at a wavelength of 580 nm of the standard sample is plotted on the abscissa and the potassium ion concentration of the standard sample is plotted on the ordinate to obtain a standard curve of the potassium ion concentration, as shown in FIG.
  • FI fluorescence intensity
  • the fluorescence intensity of the test solution find the potassium ion concentration value of the corresponding test solution on the standard curve, and divide it by 1% to obtain the potassium ion concentration value of the urine sample to be tested. The results are shown in Table 4 below.
  • the absorbance of the solution sample and the test solution at a wavelength of 590 nm was measured and recorded as .
  • the absorbances of the solution sample and the test solution at a wavelength of 590 nm and 540 nm were measured and recorded as A 2 , respectively.
  • the concentration of the sample to be tested is A 2 , the concentration of mmol/L, the actual concentration of mmol/L.
  • Example 9-16 was carried out in the background of sodium ions
  • the DNA which can be used to form the G-quadruplex in the present example is AGGGTTAGGGTTAGGGTTAGGG, and the cyanine dye used is a compound of the following formula.
  • a certain amount of DNA sample was dissolved in Tris-HCl (Tris-Na) buffer (pH 6.2) containing 20 mmol/L NaCl, and a DNA mother solution having a concentration of 500 ⁇ m ⁇ 1/ ⁇ was prepared and used.
  • Tris-Na solution with a concentration of 200mmol/L KC1
  • Tris-Na buffer to obtain potassium ions with concentrations of 0, 0.05, 0.1, 0.2. , 0.5, 1, 1.5 mmol / L of the standard sample solution.
  • the above samples were analyzed by an ultraviolet absorption spectrometer. Everything is done at room temperature without additional conditions.
  • the ultraviolet absorption spectrum collects data at a wavelength of 400 to 800 nm.
  • Example 11 The DNA capable of forming a G quadruplex used in the present example is
  • Tris-Na solution with a concentration of 200mmol/L KC1
  • Tris-Na buffer to obtain potassium ions with concentrations of 0, 0.05, 0.1, 0.2. , 0.5, 1, 1.5 mmol / L of the standard sample solution.
  • the above samples were analyzed by an ultraviolet absorption spectrometer. Everything is done at room temperature without additional conditions.
  • the ultraviolet absorption spectrum collects data at a wavelength of 400 to 800 nm.
  • the ratio of the absorbance ( ) at 560 nm of the standard sample to the absorbance (A 3 ) at 670 nm ( / A 3 ) is plotted on the abscissa, and the potassium ion concentration of the standard solution sample is plotted on the ordinate to obtain the standard of potassium ion concentration.
  • the curve is shown in Figure 7.
  • the potassium ion concentration of the corresponding test solution can be found on the standard curve, and this is divided by 1% to obtain the potassium of the urine sample to be tested.
  • the ion concentration values are shown in Table 11 below.
  • the concentration of the sample to be tested is obtained by At/A 3 mmol/L actual concentration mmol/L urine sample 1 28. 37 31. 17 Urine sample 2 36. 76 39. 71
  • the G-quadruplex can be formed in this embodiment.
  • the cyanine dye used is a compound of the formula
  • a certain amount of DNA was dissolved in Tris-HCl (Tris-Na) buffer containing 40 mmol/L NaCl to prepare a DNA mother liquor at a concentration of 3 mmol/L, and was used.
  • Tris-Na solution with a concentration of 200mmol/L KC1
  • Tris-Na buffer to obtain potassium ions with concentrations of 0, 0.05, 0.1, 0.2. , 0.5, 1, 1.5 mmol / L of the standard sample solution.
  • the above samples were analyzed by a fluorescence spectrometer. Everything is done at room temperature without additional conditions.
  • the fluorescence spectrum has an excitation wavelength of 560 nm and a wavelength collection range of 570 to 720 nm.
  • the fluorescence intensity of the standard sample at 640 nm (denoted as FI) is plotted on the abscissa and the potassium ion concentration of the standard solution sample is plotted on the ordinate to obtain a standard curve of potassium ion concentration, as shown in Fig. 8.
  • the potassium ion concentration value of the corresponding test solution can be found on the standard curve, and it is divided by 1% to obtain the potassium ion concentration value of the urine sample to be tested.
  • Table 12 The results are shown in Table 12 below.
  • the concentration of the sample to be tested is obtained by FI.
  • the concentration of mmol/L is actually the concentration of mmol/L.
  • the absorbance of the solution sample and the test solution at a wavelength of 590 nm was measured and recorded.
  • the absorbance of the solution sample and the test solution at a wavelength of 670 nm was measured and recorded as A 3 .
  • the fluorescence intensity of the solution sample and the test solution at a wavelength of 600 nm was measured and recorded as FI.
  • a certain amount of DNA sample was dissolved in Tris-HCl (Tris-Na) buffer (pH 8.0) containing 20 mmol/L NaCl, and a DNA mother solution having a concentration of ⁇ /L was prepared and used.
  • Tris-HCl Tris-Na
  • pH 8.0 Tris-Na buffer
  • the potassium ion concentration of three urine samples was verified.
  • the actual potassium ion concentration of each urine sample was as follows: urine sample 1 was 9.18 mmol/L, urine sample 2 was 32.43 mmol/L, urine sample 3 was 49.57 mmol. /L.
  • the DNA capable of forming a G-quadruplex used in the present example is AGGGTTAGGGTTAGGGTTAGGG, the cyanine dye used.
  • a certain amount of DNA sample was dissolved in Tris-HCl (Tris-Na) buffer (pH 6.2) containing 40 mmol/L NaCl to prepare a DNA mother solution at a concentration of 500 ⁇ m ⁇ 1/ ⁇ , and was used.
  • Tris-HCl Tris-Na buffer (pH 6.2) containing 40 mmol/L NaCl to prepare a DNA mother solution at a concentration of 500 ⁇ m ⁇ 1/ ⁇ , and was used.
  • the potassium ion concentration of three urine samples was verified.
  • the actual potassium ion concentration of each urine sample was as follows: urine sample 1 was 9.82 mmol/L, urine sample 2 was 20.35 mmol/L, and urine sample 3 was 78.26. Mmmol/L.
  • the DNA which can be used to form a G-quadruplex in this example is TTAGGG TTAGGGTTAGG
  • the color of the test solution was compared with the color of the standard solution sample, and it was found that the colors of the test solutions 1 and 2 were blue, and the color of the test solution 3 was purple. Therefore, the potassium ion concentration of the test solutions 1, 2, 3 is in the range of 0-60, and the potassium ion concentration of the urine samples 1, 2, 3 can be obtained in the range of 0 to 80 by dilution ratio conversion. This result is in agreement with the actual concentration of the urine sample.
  • the potassium ion concentration of three urine samples was verified.
  • the actual potassium ion concentration of each urine sample was as follows: urine sample 1 was 42.58 mmol/L, urine sample 2 was 70.65 mmol/L, and urine sample 3 was 98.34. Mmmol/L.
  • the DNA capable of forming a G-quadruplex used in the present example is AGGGTTAGGGTTAGGGTTAGGG, the cyanine dye used.
  • a certain amount of DNA sample was dissolved in Tris-HCl (Tris-Na) buffer (pH 8.2) containing 160 mmol/L NaCl to prepare a DNA mother liquor having a concentration of 500 ⁇ m ⁇ / ⁇ , and was used.
  • Tris-HCl Tris-Na buffer (pH 8.2) containing 160 mmol/L NaCl to prepare a DNA mother liquor having a concentration of 500 ⁇ m ⁇ / ⁇ , and was used.
  • Tris-Na solution with a concentration of 200mmol/L KC1
  • Tris-Na buffer to obtain potassium ions with concentrations of 0, 20, 50, 75 respectively.
  • 100, 125, 160 mmol / L standard sample solution The color of the standard solution sample is as follows: The color of the standard solution sample of 0 ⁇ 100mmol/L gradually changes from blue to purple, and the color of the standard solution of 100 ⁇ 125mmol/L gradually changes from purple to pink.
  • the urine sample in each test solution accounted for 75% of the volume of the test solution.
  • the color of the test solution was compared with the color of the standard solution sample, and it was found that the color of the test solution 1 was blue, and the color of the test solution 2 and the test solution 3 was purple. Therefore, the potassium ion concentration of the test solutions 1, 2, and 3 is in the range of 0 to 100.
  • the dilution ratio can be used to obtain the potassium ion concentration of the urine samples 1, 2, and 3 in the range of 0 to 133, and the urine sample 2, 3 potassium.
  • the ion concentration is higher than the urine sample 1 potassium ion concentration. This result is in agreement with the actual concentration of the urine sample.
  • a certain amount of DNA sample was dissolved in Tris-HCl (Tris-Na) buffer (pH 7.0) containing 20 mmol/L NaCl to prepare a DNA mother solution having a concentration of 20 (mol/L), which was used.
  • Tris-HCl Tris-Na buffer (pH 7.0) containing 20 mmol/L NaCl to prepare a DNA mother solution having a concentration of 20 (mol/L), which was used.
  • the color of the test solution was compared with the color of the standard solution sample, and it was found that the color of the test solution 1 was blue, the test solution 2 was purple, and the color of the test solution 3 was pink. Therefore, the potassium ion concentration of the test solution 1 is in the range of 0 to 10, and the potassium ion concentration of the test solution 2 and the test solution 3 is in the range of 10 20 , and the potassium ion concentration of the urine sample 1 can be obtained from 0 to 20 by dilution ratio conversion.
  • the range of urine sample 2 and urine sample 3 has a potassium ion concentration in the range of 20 to 40. This result is in agreement with the actual concentration of the urine sample.
  • the potassium ion concentration of three urine samples was verified.
  • the actual potassium ion concentration of each urine sample was as follows: urine sample 1 was 85.32 mmol/L, urine sample 2 was 100.54 mmol/L, urine sample 3 was 126.32 mmol. /L, the DNA used is GGGCGCGGGAGGAAGGGGGCGGG, and the cyanine dye used is a compound of the formula:
  • a certain amount of DNA sample was dissolved in Tris-HCl (Tris-Na) buffer (pH 6.2) containing 300 mmol/L NaCl to prepare a DNA mother liquor having a concentration of 20 (mol/L), which was used.
  • Tris-HCl Tris-Na buffer (pH 6.2) containing 300 mmol/L NaCl to prepare a DNA mother liquor having a concentration of 20 (mol/L), which was used.
  • the color of the test solution was compared with the color of the standard solution sample, and it was found that the colors of the test solutions 1, 2, and 3 were blue. Therefore, the potassium ion concentration of the test solutions 1, 2, 3 is in the range of 0 150, and the potassium ion concentration of the three urine samples is in the range of 0 300 by the dilution ratio conversion. This result is in agreement with the actual concentration of the urine sample.
  • a certain amount of DNA sample was dissolved in Tris-HCl (Tris-Na) buffer ( ⁇ 8.0) containing 10 mmol/L NaCl to prepare a DNA mother solution having a concentration of 200 ⁇ m ⁇ 1/ ⁇ , and was used.
  • test solution In the other three samples, 200 ⁇ of the urine sample to be tested was added to obtain three test solutions, and the urine sample in each test solution accounted for 17% of the volume of the test solution.
  • the color of the test solution was compared with the color of the standard solution sample, and it was found that the color of the test solution 1 was blue, and the color of the test solution 2 and the test solution 3 was pink. Therefore, the potassium ion concentration of the test solution 1 is in the range of 0-6, and the potassium ion concentration of the test solution 2 and the test solution 3 is in the range of 6-10, and the potassium ion concentration of the urine sample 1 can be obtained by the dilution ratio conversion. In the range of 35, the potassium ion concentration of urine sample 2 and urine sample 3 is in the range of 35 to 59. This result is in agreement with the actual concentration of the urine sample.
  • pH (pH 6.2) a DNA stock solution of ⁇ /L was prepared and used.
  • Tris-Na solution with a concentration of 200mmol/L KC1
  • Tris-Na buffer to obtain potassium ions with concentrations of 0, 10, 20, respectively. 30, 40, 50, 60 mmol/L standard sample solution.
  • the color of the standard solution sample is as follows: The color of the standard solution sample of 0 ⁇ 30mmol/L gradually changes from blue to purple, and the color of the standard solution of 30 ⁇ 60mmol/L gradually changes from purple to pink.
  • the color of the test solution 1 was found. In blue, the color of Test Solution 2 and Test Solution 3 is purple. Therefore, the potassium ion concentration of the test solution 1 is in the range of 0-30, and the potassium ion concentration of the test solution 2 and the test solution 3 is in the range of 30 60.
  • the potassium ion concentration of the urine sample 1 can be obtained by the dilution ratio conversion from 0 to 45.
  • the range of urine sample 2 and urine sample 3 has a potassium ion concentration in the range of 45 to 91. This result is in agreement with the actual concentration of the urine sample.
  • One of the remarkable features of the present invention is: detection of changes in DNA conformation based on potassium ions, potassium ions cause DNA conformational changes, and DNA conformational changes cause changes in the aggregation pattern of cyanine dyes, resulting in solution color or absorption, fluorescence The spectrum changes.
  • the system is simple in composition and simple in reaction. Potassium ion is the "initiator" of the whole reaction, which ensures the accuracy of the detection.
  • the third characteristic feature of the present invention is that the use of a cyanine dye supramolecular probe has high reaction sensitivity and color change, and can be visually observed.

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Abstract

La présente invention concerne un procédé de détection d'une concentration en ion potassium dans un échantillon liquide, comprenant les étapes consistant à : (1) formuler une pluralité d'échantillons en solution avec différentes concentrations en ion potassium avec une solution tampon à un pH de 6,2-8,2, chacun des échantillons en solution contenant des molécules d'ADN pouvant former des G-quadruplexes à la même concentration et un colorant cyanine à la même concentration ; (2) placer la pluralité d'échantillons en solution sous un spectromètre ou spectrophotomètre d'absorption dans l'ultraviolet/le visible, et détecter la valeur d'absorbance à une première longueur d'onde et la valeur d'absorbance à une deuxième longueur d'onde, ou placer la pluralité d'échantillons en solution sous un spectrophotomètre de fluorescence, et détecter la valeur d'intensité de fluorescence à une troisième longueur d'onde avec une longueur d'onde d'excitation de 560 nm, la première longueur d'onde étant la plage allant de 560 nm à 590 nm, la deuxième longueur d'onde étant dans la plage allant de 500 nm à 540 nm, et la troisième longueur d'onde étant dans la plage allant de 580 nm à 640 nm ; (3) représenter graphiquement la concentration en ion potassium de chacun des échantillons en solution en abscisse ou en ordonnée et la valeur d'absorbance à la première longueur d'onde ou la valeur d'absorbance à la deuxième longueur d'onde ou le rapport entre la valeur d'absorbance à la première longueur d'onde et la valeur d'absorbance à la deuxième longueur d'onde, ou la valeur d'intensité de fluorescence à la troisième longueur d'onde mesurée dans l'étapes (2) en ordonnée ou en abscisse, afin d'obtenir une courbe étalon de la concentration en ion potassium ; (4) ajouter des molécules d'ADN pouvant former des G-quadruplexes, un composé de formule I et une solution tampon dans un échantillon liquide à analyser, afin de permettre que les molécules d'ADN pouvant former des G-quadruplexes, le composé de formule I et la valeur de pH dans l'échantillon liquide à analyser correspondent à ceux des échantillons en solution dans l'étape (1), afin d'obtenir une solution d'essai ; (5) placer la solution d'essai obtenue dans l'étape (4) sous le spectromètre ou spectrophotomètre d'absorption dans l'ultraviolet/le visible et détecter les valeurs d'absorbance de la solution d'essai à la première longueur d'onde et à la deuxième longueur d'onde, ou placer la solution d'essai sous le spectrophotomètre de fluorescence, et détecter la valeur d'intensité de fluorescence à une troisième longueur d'onde avec une longueur d'onde d'excitation de 560 nm; (6) déterminer la concentration en ion potassium correspondante de la solution d'essai dans la courbe étalon de la concentration en ion potassium obtenue dans l'étape (3) en utilisant la valeur d'absorbance à la première longueur d'onde ou la valeur d'absorbance à la deuxième longueur d'onde, ou le rapport entre la valeur d'absorbance à la première longueur d'onde et la valeur d'absorbance à la deuxième longueur d'onde ou la valeur d'intensité de fluorescence à la troisième longueur d'onde mesurées dans l'étape (5), puis calculer la concentration en ion potassium de l'échantillon à analyser par le rapport de dilution de l'échantillon à analyser.
PCT/CN2013/077335 2012-06-18 2013-06-17 Procédé, système et kit de détection de la concentration en ion potassium WO2013189265A1 (fr)

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CN201210206227.2A CN102735664B (zh) 2012-06-18 2012-06-18 钾离子浓度检测方法
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CN201210205861.4A CN102866149B (zh) 2012-06-18 2012-06-18 钾离子浓度检测试剂盒
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CN114112955A (zh) * 2021-11-23 2022-03-01 福州大学 一种测量溶液中ez宽度的方法

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