WO2022099972A1 - Relaxation nuclear magnetic resonance method for measuring glucose content in liquid biological sample - Google Patents
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 69
- 239000008103 glucose Substances 0.000 title claims abstract description 69
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- 239000007788 liquid Substances 0.000 title claims abstract description 34
- 238000001225 nuclear magnetic resonance method Methods 0.000 title claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 38
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- 238000005481 NMR spectroscopy Methods 0.000 claims abstract description 29
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- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 15
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- 102000006395 Globulins Human genes 0.000 claims description 2
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- 108010063954 Mucins Proteins 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
- G01N24/082—Measurement of solid, liquid or gas content
Definitions
- the invention belongs to the technical field of biochemical method analysis and detection, and in particular relates to a relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample.
- relaxation NMR technology has been widely used in food science, biological detection and other fields.
- it is not sensitive to the weak changes in the content of small molecules in the sample. Specifically, it is used in liquids.
- the glucose level in biological samples such as blood, saliva, and urine is generally low, only 0-30mM, it is difficult to accurately measure its content by direct relaxation nuclear magnetic resonance detection, so it is necessary to design a method. To expand the detectable amount due to different glucose levels in the sample.
- MNPs modified magnetic nanoparticles
- Fe 3+ /Fe 2+ in aqueous solution can form water ions, and in the same concentration of aqueous solution, the T 1 of Fe 2+ is higher than that of Fe 3+ .
- there are various types of redox reactions other than glucose in biological samples which can also cause the relaxation transformation between Fe 3+ and Fe 2+ , which greatly interferes with the specificity of this detection method.
- the purpose of the present invention is to further expand the application of relaxation nuclear magnetic resonance technology in the direction of biological detection, and propose a relaxation nuclear magnetic resonance method for detecting the glucose content of liquid biological samples, more specifically, it is based on glucose oxidase and acidification.
- Potassium permanganate was used as the reaction substrate, and organic reagents were used to remove the original protein interference in biological samples.
- the changes in longitudinal relaxation time of potassium permanganate were detected by 1 H-NMR relaxation to calculate the glucose content in the tested samples.
- a relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample of the present invention is realized by the following technical scheme:
- Glucose oxidase and acidified potassium permanganate were used as reaction substrates, and organic reagents were used to remove the original protein interference in biological samples, and the change of potassium permanganate relaxation time was detected by 1 H-NMR relaxation to calculate the sample to be tested.
- Glucose content the method specifically comprises the steps:
- Step a Add anhydrous ethanol or other organic reagents with the same effect to the liquid biological sample to be tested, mix well and let stand to denature and precipitate large proteins in the sample: the liquid biological sample is pre-cooled in advance, and then the same pre-cooled Absolute ethanol or other organic reagents, fully invert and mix, and let stand at pre-cooling temperature to wait for the denatured protein to fully precipitate;
- Step b Evaporate the residual organic reagent from the supernatant: centrifuge the precipitated sample solution, take out the separated supernatant, divide it into two equal parts and record them as groups A and B, and place them in a drying box to fully evaporate;
- Step c Add standard detection solution and control group blank solution to group A and B respectively to react: group A is added with an acidic solution containing glucose oxidase and potassium permanganate, and group B of blank control group is added with permanganic acid containing only equal concentration Potassium acid solution, mix separately;
- Step d Detect the difference between the relaxation times of groups A and B with a nuclear magnetic resonance instrument: respectively sample the groups A and B after the final reaction is completed, and perform nuclear magnetic resonance detection of their relaxation times T 1 , T 2 or other parameters that can characterize the relaxation of the sample. The measured quantity, the difference between the relaxation times of the two groups is recorded as ⁇ T;
- Step e Comparing the measured relaxation time difference with the known data model of the sample type to obtain the glucose content of the tested biological sample: Substitute the A and B groups ⁇ T of the tested liquid biological sample as the input into the known data model, and the resulting output is the measured glucose content.
- the liquid biological samples to be tested include whole blood, serum, urine, saliva, and biologically derived samples containing glucose and having a liquid texture, including albumin, globulin, and mucin that interfere with the detection. Denaturation occurs in the solvent, resulting in a flocculent or agglomerated precipitate.
- the evaporation residue is to leave the liquid sample open in a drying oven until the organic solvent evaporates completely in an environment above its boiling point.
- the standard detection solution is to dissolve solid glucose oxidase and potassium permanganate in the acetic acid buffer of pH 5.
- the content of the glucose oxidase varies according to the magnitude of the glucose content in the biological sample to be tested; the content of the potassium permanganate varies according to the upper limit of detection required by the biological sample to be tested. the difference.
- the blank solution of the control group is that potassium permanganate is dissolved in the acetic acid buffer of pH 5, and the final concentration is consistent with the potassium permanganate concentration in the standard detection solution.
- the nuclear magnetic resonance instrument is a low-field nuclear magnetic resonance instrument that meets the requirements for measuring the 1 H-NMR relaxation signal, and the sample size for the sampling measurement varies according to the requirements of the instrument.
- the relaxation time is measured using an inversion recovery pulse sequence IR or a multi-echo measurement sequence CPMG.
- the known data model is a series of ⁇ T obtained after the above steps are performed on the specified biological samples whose basic components are consistent with the tested samples, that is, both belong to serum, urine or saliva, and whose glucose content is known.
- the data model obtained by the simultaneous glucose concentration, each type of biological sample has a different data model corresponding to it.
- the detection instrument of the sample to be tested should be consistent with the relaxation nuclear magnetic resonance detection instrument used to establish the data model, with the same proton resonance frequency, test temperature, and a series of measurements using the same measurement pulse sequence, scan times, and echo interval. parameter.
- the biological samples whose glucose concentration has been calibrated are based on the same components as the tested samples, such as serum, urine or saliva, each type of biological sample has a different data model corresponding to it, and its glucose content is known.
- the biochemical reaction involved in the present invention is that glucose is hydrolyzed under the action of glucose oxidase to generate hydrogen peroxide, and potassium permanganate is oxidized, wherein the change of the state of metal ions causes the change of the relaxation characteristics of the solution.
- the reaction equation as follows:
- the essence of metal ions affecting the solution relaxation rate is that the local field generated by the unpaired electron spins of paramagnetic metals affects the uniformity of the static magnetic field, while the essence of metal ion valence changes is its magnetic moment, ionic radius and electron spin relaxation. Therefore, whenever a reaction brings about a change in the valence of the metal ion, it results in a corresponding change in the relaxation rate of the solution, which can be quantified by 1H-NMR relaxation measurements.
- the relaxation nuclear magnetic resonance method of the present invention effectively amplifies the relaxation change of the sample caused by the difference of the glucose content by introducing metal ions. It is more suitable for very low concentrations of glucose in the range of 0-30mM in biological samples.
- the 1H-NMR method used in the present invention is to measure the glucose content by mixing biological samples with organic reagents to precipitate irrelevant components, adding glucose oxidase and acidified potassium permanganate to the supernatant, and then performing NMR relaxation measurement. Compared with the direct detection of glucose content by relaxation, the detection ability at low glucose concentration is greatly improved.
- the invention provides a method for detecting glucose content in a liquid biological sample by nuclear magnetic resonance relaxation method.
- the introduced reactant has a weak effect on the glucose content.
- the change is more sensitive, that is, the redox reaction of glucose oxidase-acidified potassium permanganate is used, the change of relaxation time is measured by NMR, and then the glucose concentration in the original sample is calculated by the data model.
- the NMR relaxation method adopted in the present invention has high efficiency and low detection limit, and is more suitable for the detection of low glucose content in biological samples.
- FIG. 1 is a process flow chart of the present invention for detecting glucose content in a liquid biological sample.
- Fig. 2 is an example of the data model of the comparison between ⁇ T 1 and glucose concentration of the present invention, showing the difference ⁇ T 1 of longitudinal relaxation time and glucose concentration plotted by calibration of a sample of bovine serum albumin solution with known glucose concentration in Example 1 comparison model.
- the present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.
- the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
- step 2 The supernatant obtained by centrifugation in step 1 was divided into groups A and B to take 150uL each, and placed in a drying oven at 85°C to evaporate to completeness;
- step 3 To the dried products A and B groups obtained in step 2, add 1 mL of the standard detection solution and the standard blank solution with the same proportion of the data model corresponding to the BSA solution sample respectively, shake to dissolve and mix, and let stand for 1 hour to make it fully reaction;
- step 4 Take 200uL of the samples after the reaction obtained in step 3 and put them into a 7.5mm nuclear magnetic resonance test tube. Use a Bruker mq60 low-field nuclear magnetic resonance measuring instrument. The measurement pulse is IR, the number of scans is 8, and the number of sampling points is 21. relaxation time measurement;
- the preparation of the standard detection solution and the standard blank solution is taken as an example for the detection of samples with a glucose concentration range of 1-20 mM.
- step 3 Measure 2mL of the glucose oxidase solution in step 2, add 40uL of potassium permanganate solution in step 1, invert and mix, take 1mL of it and dilute it to 20mL with acetic acid buffer of pH 5 to obtain a standard detection solution;
- step 4 Measure 20uL potassium permanganate solution of step 1, dilute to 20mL with the acetic acid buffer of pH 5, invert and mix to obtain standard blank solution.
- the glucose concentration in step 2 is 100mM BSA solution, dilute to 4mL with the BSA solution in step 1, and obtain glucose concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20mM BSA solution, pre-cooled at 4°C;
- the supernatant obtained by centrifugation in step 4 was divided into groups A and B to take 150uL each, and placed in a drying box at 85°C to evaporate to completeness.
- step 7 Take 200uL of the samples after the reaction obtained in step 6 and put them into a 7.5mm nuclear magnetic resonance test tube. Use a Bruker mq60 low-field nuclear magnetic resonance measuring instrument. The measurement pulse is IR, the number of scans is 8 times, and the number of sampling points is 21. relaxation time measurement;
- the above-mentioned embodiment is an embodiment of the present invention for the bovine serum albumin solution sample, but the embodiment of the present invention is not limited by the above-mentioned example, and any other changes made without departing from the spirit and principle of the present invention, Modifications, substitutions, combinations, and simplifications should all be equivalent substitutions, which are all included within the protection scope of the present invention.
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Abstract
A relaxation nuclear magnetic resonance method for measuring glucose content in a liquid biological sample, wherein glucose oxidase and acidified potassium permanganate are used as reaction substrates, and the glucose content in a measured sample can be calculated by detecting the change of relaxation time by means of 1H-NMR. The method specifically comprises: step a: adding absolute ethyl alcohol or other equivalent organic reagents to a sample being measured in a certain proportion, mixing them well and letting the mixture stand still to denature and precipitate interfering proteins in the sample; step b: taking the supernatant and evaporating residual organic reagents; step c: respectively adding a detection solution and a control solution to a group A and a group B for reaction; step d: measuring a relaxation difference between the groups A and B by using a nuclear magnetic resonance instrument; and step e: calculating the glucose content in the measured sample according to the measured relaxation time difference and a known model under the sample category. By means of the relaxation nuclear magnetic resonance method, the glucose content in various liquid biological samples can be effectively measured, helping to further study the application of nuclear magnetic resonance technology in the direction of biological sugar detection.
Description
本发明属于生物化学方法分析检测技术领域，具体涉及一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法。The invention belongs to the technical field of biochemical method analysis and detection, and in particular relates to a relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample.
近年来弛豫核磁共振技术被广泛应用于食品科学、生物检测等领域，但受限于弛豫核磁共振分辨率较低，对样本中小分子含量的微弱变化并不敏感，具体地，应用于液体生物样本中葡萄糖含量的检测时，由于生物样本如血液、唾液、尿液中的葡萄糖水平一般较低，仅有0-30mM，直接进行弛豫核磁共振检测难以准确测定其含量，因此需要设计方法扩大样本中葡萄糖含量不同引起的可检出量。In recent years, relaxation NMR technology has been widely used in food science, biological detection and other fields. However, due to the low resolution of relaxation NMR, it is not sensitive to the weak changes in the content of small molecules in the sample. Specifically, it is used in liquids. In the detection of glucose content in biological samples, since the glucose level in biological samples such as blood, saliva, and urine is generally low, only 0-30mM, it is difficult to accurately measure its content by direct relaxation nuclear magnetic resonance detection, so it is necessary to design a method. To expand the detectable amount due to different glucose levels in the sample.
在本领域中已知的一种检测方法是基于目标诱导的MNP聚集的磁弛豫转换分析，通过经修饰的磁性纳米颗粒(MNP)的特异性吸附，放大葡萄糖引起的液体样本弛豫变化。但在复杂的生物样本中，由于MNP易发生非特异性吸附，不受控制的聚集会严重影响测定的准确性。在此基础上，本领域已提出的另一种解决方法是无MNPs磁性测定法的开发。水溶液中的Fe
3+/Fe
2+可以形成水离子，并且在同浓度的水溶液中，Fe
2+的T
1高于Fe
3+。然而生物样本中除葡萄糖以外的其他物质也存在各种不同类型的氧化还原反应，同样会引起Fe
3+和Fe
2+之间的弛豫转化，极大干扰此检测方法的特异性。
One detection method known in the art is a magnetic relaxation transition analysis based on target-induced aggregation of MNPs, which amplifies glucose-induced relaxation changes in liquid samples through specific adsorption of modified magnetic nanoparticles (MNPs). However, in complex biological samples, uncontrolled aggregation can seriously affect the accuracy of the assay because MNPs are prone to nonspecific adsorption. On this basis, another solution that has been proposed in the art is the development of MNPs-free magnetic assays. Fe 3+ /Fe 2+ in aqueous solution can form water ions, and in the same concentration of aqueous solution, the T 1 of Fe 2+ is higher than that of Fe 3+ . However, there are various types of redox reactions other than glucose in biological samples, which can also cause the relaxation transformation between Fe 3+ and Fe 2+ , which greatly interferes with the specificity of this detection method.
葡萄糖氧化酶与葡萄糖的反应具有高度特异性，而高锰酸钾作为反应产物过氧化氢常用的指示剂，目前还未见其在葡萄糖含量检测中的相关报道。The reaction between glucose oxidase and glucose is highly specific, and potassium permanganate, as a commonly used indicator of the reaction product hydrogen peroxide, has not yet been reported in the detection of glucose content.
发明内容SUMMARY OF THE INVENTION
技术问题：本发明的目的在于进一步拓展弛豫核磁共振技术在生物检测方向上的应用，提出一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，更具体地，是以葡萄糖氧化酶和酸化高锰酸钾作为反应底物，并利用有机试剂除去生物样本中原有的蛋白质干扰，通过
1H-NMR弛豫检测高锰酸钾纵向弛豫时间的变化来计算被测样本中葡萄糖含量。
Technical problem: The purpose of the present invention is to further expand the application of relaxation nuclear magnetic resonance technology in the direction of biological detection, and propose a relaxation nuclear magnetic resonance method for detecting the glucose content of liquid biological samples, more specifically, it is based on glucose oxidase and acidification. Potassium permanganate was used as the reaction substrate, and organic reagents were used to remove the original protein interference in biological samples. The changes in longitudinal relaxation time of potassium permanganate were detected by 1 H-NMR relaxation to calculate the glucose content in the tested samples.
技术方案：本发明的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法通过如下技术方案予以实现：Technical scheme: A relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample of the present invention is realized by the following technical scheme:
以葡萄糖氧化酶和酸化高锰酸钾作为反应底物，并利用有机试剂除去生物样本中原有的蛋白质干扰，通过
1H-NMR弛豫检测高锰酸钾弛豫时间的变化来计算被测样本中葡萄糖含量，该方法具体包括如下步骤：
Glucose oxidase and acidified potassium permanganate were used as reaction substrates, and organic reagents were used to remove the original protein interference in biological samples, and the change of potassium permanganate relaxation time was detected by 1 H-NMR relaxation to calculate the sample to be tested. Glucose content, the method specifically comprises the steps:
步骤a.将被测液体生物样本加入无水乙醇或其他具有相同效果的有机试剂混匀后静置，使样本中大蛋白变性沉淀：液体生物样本事先进行预冷，随后加入 同样经预冷的无水乙醇或其他有机试剂，充分翻转混匀后在预冷温度下静置，等待发生变性的蛋白质充分沉淀；Step a. Add anhydrous ethanol or other organic reagents with the same effect to the liquid biological sample to be tested, mix well and let stand to denature and precipitate large proteins in the sample: the liquid biological sample is pre-cooled in advance, and then the same pre-cooled Absolute ethanol or other organic reagents, fully invert and mix, and let stand at pre-cooling temperature to wait for the denatured protein to fully precipitate;
步骤b.取上层清液蒸发残留有机试剂：将产生沉淀的样本液进行离心，取出分离的上清液，分为等量的两份记为A、B组，置于干燥箱充分蒸发；Step b. Evaporate the residual organic reagent from the supernatant: centrifuge the precipitated sample solution, take out the separated supernatant, divide it into two equal parts and record them as groups A and B, and place them in a drying box to fully evaporate;
步骤c.将A、B组分别加入标准检测溶液及对照组空白溶液进行反应：A组加入含有葡萄糖氧化酶及高锰酸钾的酸性溶液，空白对照组B组加入只含有等浓度高锰酸钾的酸性溶液，分别混匀；Step c. Add standard detection solution and control group blank solution to group A and B respectively to react: group A is added with an acidic solution containing glucose oxidase and potassium permanganate, and group B of blank control group is added with permanganic acid containing only equal concentration Potassium acid solution, mix separately;
步骤d.用核磁共振仪器检测A、B组弛豫时间之差：将最终反应完成的A、B组分别取样进行核磁共振检测其弛豫时间T
1、T
2或其他可表征样本弛豫的测量量，两组弛豫时间的差值记为ΔT；
Step d. Detect the difference between the relaxation times of groups A and B with a nuclear magnetic resonance instrument: respectively sample the groups A and B after the final reaction is completed, and perform nuclear magnetic resonance detection of their relaxation times T 1 , T 2 or other parameters that can characterize the relaxation of the sample. The measured quantity, the difference between the relaxation times of the two groups is recorded as ΔT;
步骤e.根据测得的弛豫时间差与该样本种类已知的数据模型对照得出被测生物样本的葡萄糖含量：将被测液体生物样本的A、B组ΔT作为输入量代入已知的数据模型，得出的输出量即为测得的葡萄糖含量。Step e. Comparing the measured relaxation time difference with the known data model of the sample type to obtain the glucose content of the tested biological sample: Substitute the A and B groups ΔT of the tested liquid biological sample as the input into the known data model, and the resulting output is the measured glucose content.
其中：in:
所述被测液体生物样本包括全血、血清、尿液、唾液及含有葡萄糖且质地为液体的生物来源类样本，其中具有一些干扰检测的白蛋白、球蛋白、粘蛋白，在特定浓度的有机溶剂中发生变性，产生絮状或团块状沉淀物。The liquid biological samples to be tested include whole blood, serum, urine, saliva, and biologically derived samples containing glucose and having a liquid texture, including albumin, globulin, and mucin that interfere with the detection. Denaturation occurs in the solvent, resulting in a flocculent or agglomerated precipitate.
所述蒸发残留是将液体样本敞口置于干燥箱中直至其中有机溶剂在高于其沸点的环境中蒸发完全。The evaporation residue is to leave the liquid sample open in a drying oven until the organic solvent evaporates completely in an environment above its boiling point.
所述标准检测溶液是将固体葡萄糖氧化酶及高锰酸钾溶解于pH 5的乙酸缓冲液中。The standard detection solution is to dissolve solid glucose oxidase and potassium permanganate in the acetic acid buffer of pH 5.
所述的葡萄糖氧化酶，其含量根据被测生物样本中葡萄糖含量的量级不同而有所区别；所述的高锰酸钾，其含量根据被测生物样本所需的检测上限不同而有所区别。The content of the glucose oxidase varies according to the magnitude of the glucose content in the biological sample to be tested; the content of the potassium permanganate varies according to the upper limit of detection required by the biological sample to be tested. the difference.
所述对照组空白溶液是将高锰酸钾溶解于pH 5的乙酸缓冲液中，终浓度与标准检测溶液中的高锰酸钾浓度一致。The blank solution of the control group is that potassium permanganate is dissolved in the acetic acid buffer of pH 5, and the final concentration is consistent with the potassium permanganate concentration in the standard detection solution.
所述核磁共振仪器为满足
1H-NMR弛豫信号测量要求的低场核磁共振仪器，所述取样测量的样本量根据仪器要求有所不同。
The nuclear magnetic resonance instrument is a low-field nuclear magnetic resonance instrument that meets the requirements for measuring the 1 H-NMR relaxation signal, and the sample size for the sampling measurement varies according to the requirements of the instrument.
所述弛豫时间利用反转回复脉冲序列IR或多回波测量序列CPMG测得。The relaxation time is measured using an inversion recovery pulse sequence IR or a multi-echo measurement sequence CPMG.
所述已知的数据模型是对基础成分与被测样本一致，即同属血清、尿液或唾液，并且其葡萄糖含量已知的指定生物样本进行如上步骤的检测后获得的一系列ΔT与对应的葡萄糖浓度联立得到的数据模型，每类生物样本都具有与之对应的不同数据模型。The known data model is a series of ΔT obtained after the above steps are performed on the specified biological samples whose basic components are consistent with the tested samples, that is, both belong to serum, urine or saliva, and whose glucose content is known. The data model obtained by the simultaneous glucose concentration, each type of biological sample has a different data model corresponding to it.
所述被测样本的检测仪器需与建立数据模型使用的弛豫核磁共振检测仪器一致，具有相同的质子共振频率、测试温度，使用相同的测量脉冲序列、扫描次数、回波间隔的一系列测量参数。The detection instrument of the sample to be tested should be consistent with the relaxation nuclear magnetic resonance detection instrument used to establish the data model, with the same proton resonance frequency, test temperature, and a series of measurements using the same measurement pulse sequence, scan times, and echo interval. parameter.
所述已标定葡萄糖浓度的生物样本为基础成分与被测样本一致，如同属血清、尿液或唾液，每类生物样本都具有与之对应的不同数据模型，并且其葡萄糖含量已知。The biological samples whose glucose concentration has been calibrated are based on the same components as the tested samples, such as serum, urine or saliva, each type of biological sample has a different data model corresponding to it, and its glucose content is known.
本发明中涉及的生物化学反应，具体地说，是葡萄糖在葡萄糖氧化酶作用下水解产生过氧化氢，使高锰酸钾氧化，其中金属离子状态的改变引起溶液弛豫特性的变化，反应方程如下：The biochemical reaction involved in the present invention, specifically, is that glucose is hydrolyzed under the action of glucose oxidase to generate hydrogen peroxide, and potassium permanganate is oxidized, wherein the change of the state of metal ions causes the change of the relaxation characteristics of the solution. The reaction equation as follows:
C
6H
12O
6+O
2+H
2O→C
6H
12O
7+H
2O
2
C 6 H 12 O 6 +O 2 +H 2 O→C 6 H 12 O 7 +H 2 O 2
2KMnO
4+5H
2O
2+6H
+→2Mn
2++2K
++5O
2+8H
2O
2KMnO 4 +5H 2 O 2 +6H + →2Mn 2+ +2K + +5O 2 +8H 2 O
金属离子影响溶液弛豫速率的本质是顺磁性金属的未配对电子自旋产生的局域场影响静磁场均匀性，而金属离子价性改变的实质是其磁矩、离子半径和电子自旋弛豫的变化，因此只要反应带来金属离子的价性改变，就相应导致溶液弛豫速率的变化，这种变化可以通过1H-NMR弛豫测量量化。The essence of metal ions affecting the solution relaxation rate is that the local field generated by the unpaired electron spins of paramagnetic metals affects the uniformity of the static magnetic field, while the essence of metal ion valence changes is its magnetic moment, ionic radius and electron spin relaxation. Therefore, whenever a reaction brings about a change in the valence of the metal ion, it results in a corresponding change in the relaxation rate of the solution, which can be quantified by 1H-NMR relaxation measurements.
本发明的弛豫核磁共振检测液体生物样本葡萄糖含量的方法，与现有测定液体样本的弛豫核磁共振技术相比，通过引入金属离子，有效放大了样本随葡萄糖含量不同引起的弛豫变化，对生物样本中0-30mM范围极低浓度的葡萄糖含量更加适用。Compared with the existing relaxation nuclear magnetic resonance technology for measuring the liquid biological sample, the relaxation nuclear magnetic resonance method of the present invention effectively amplifies the relaxation change of the sample caused by the difference of the glucose content by introducing metal ions. It is more suitable for very low concentrations of glucose in the range of 0-30mM in biological samples.
本发明采用的1H-NMR法测定葡萄糖含量的方法，是将生物样本掺入有机试剂沉淀无关组分，清液加入葡萄糖氧化酶及酸化高锰酸钾反应后进行NMR弛豫测量，与常规NMR弛豫直接检测葡萄糖含量相比，极大提高了低葡萄糖浓度下的检测能力。The 1H-NMR method used in the present invention is to measure the glucose content by mixing biological samples with organic reagents to precipitate irrelevant components, adding glucose oxidase and acidified potassium permanganate to the supernatant, and then performing NMR relaxation measurement. Compared with the direct detection of glucose content by relaxation, the detection ability at low glucose concentration is greatly improved.
有益效果：本发明与现有技术相比，具有如下有益效果：Beneficial effects: compared with the prior art, the present invention has the following beneficial effects:
本发明提供了一种通过核磁共振弛豫法检测液体生物样本中葡萄糖含量的方法，与现有检测液体样本的弛豫核磁共振技术相比，通过引入的反应物，本发明对葡萄糖含量的微弱变化更为敏感，即采用葡萄糖氧化酶-酸化高锰酸钾的氧化还原反应，通过NMR测定出弛豫时间的变化，进而通过数据模型计算出原样本中的葡萄糖浓度。本发明采用的NMR弛豫法高效、检出限低，更适用于生物样本中低葡萄糖含量的检测。The invention provides a method for detecting glucose content in a liquid biological sample by nuclear magnetic resonance relaxation method. Compared with the existing relaxation nuclear magnetic resonance technology for detecting liquid samples, the introduced reactant has a weak effect on the glucose content. The change is more sensitive, that is, the redox reaction of glucose oxidase-acidified potassium permanganate is used, the change of relaxation time is measured by NMR, and then the glucose concentration in the original sample is calculated by the data model. The NMR relaxation method adopted in the present invention has high efficiency and low detection limit, and is more suitable for the detection of low glucose content in biological samples.
图1为本发明液体生物样本中葡萄糖含量检测的工艺流程图。FIG. 1 is a process flow chart of the present invention for detecting glucose content in a liquid biological sample.
图2为本发明的ΔT
1与葡萄糖浓度对照的数据模型示例，显示了实施例1中用已知葡萄糖浓度的牛血清白蛋白溶液样本定标绘制的纵向弛豫时间之差ΔT
1与葡萄糖浓度的对照模型。
Fig. 2 is an example of the data model of the comparison between ΔT 1 and glucose concentration of the present invention, showing the difference ΔT 1 of longitudinal relaxation time and glucose concentration plotted by calibration of a sample of bovine serum albumin solution with known glucose concentration in Example 1 comparison model.
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备均为本技术领域常规试剂、方法和设备。The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
除非特别说明，以下实施例所用试剂和材料均为市购。Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
实施例1Example 1
检测BSA溶液样本，其葡萄糖含量未知。A sample of BSA solution was tested and its glucose content was unknown.
1.量取样本500uL，放置于4℃预冷，加入1000uL同样在4℃预冷的无水乙醇，翻转混匀，放置于4℃沉淀20分钟，室温离心(3500转/分钟，10分钟)；1. Measure 500uL of the sample, place it at 4°C to pre-cool, add 1000uL of anhydrous ethanol also pre-cooled at 4°C, invert and mix well, place it at 4°C for 20 minutes, and centrifuge at room temperature (3500 rpm, 10 minutes) ;
2.将步骤一离心所得上清液，分A、B组各取150uL，置于干燥箱85℃蒸发至完全；2. The supernatant obtained by centrifugation in step 1 was divided into groups A and B to take 150uL each, and placed in a drying oven at 85°C to evaporate to completeness;
3.对步骤二所得的干燥物A、B组，分别加入与BSA溶液样本对应的数据模型同配比的标准检测溶液及标准空白溶液各1mL，振荡溶解混匀，静置1小时使其充分反应；3. To the dried products A and B groups obtained in step 2, add 1 mL of the standard detection solution and the standard blank solution with the same proportion of the data model corresponding to the BSA solution sample respectively, shake to dissolve and mix, and let stand for 1 hour to make it fully reaction;
4.将步骤三所得的反应完成后的样本各取200uL装入7.5mm核磁共振测试管，使用Bruker mq60低场核磁共振测量仪，测量脉冲为IR，扫描次数8次，采样点数21，进行纵向弛豫时间测量；4. Take 200uL of the samples after the reaction obtained in step 3 and put them into a 7.5mm nuclear magnetic resonance test tube. Use a Bruker mq60 low-field nuclear magnetic resonance measuring instrument. The measurement pulse is IR, the number of scans is 8, and the number of sampling points is 21. relaxation time measurement;
5.将步骤四得到的样本A、B组间的纵向弛豫时间之差ΔT
1代入BSA溶液样本及步骤四使用的核磁共振仪器对应的数据模型，得到与该ΔT
1对应的葡萄糖浓度值，即为被测样本的葡萄糖浓度。
5. Substitute the longitudinal relaxation time difference ΔT 1 between the samples A and B groups obtained in step 4 into the data model corresponding to the BSA solution sample and the nuclear magnetic resonance instrument used in step 4 to obtain the glucose concentration value corresponding to this ΔT 1 , is the glucose concentration of the tested sample.
实施例2Example 2
标准检测溶液及标准空白溶液的制作，以检测葡萄糖浓度范围1-20mM的样本为例。The preparation of the standard detection solution and the standard blank solution is taken as an example for the detection of samples with a glucose concentration range of 1-20 mM.
1.根据反应方程计算所需的高锰酸钾低限浓度并适量放大。称取0.15g高锰酸钾，室温溶解于4mL pH 5的乙酸缓冲液中；1. Calculate the required minimum concentration of potassium permanganate according to the reaction equation and enlarge it appropriately. Weigh 0.15g potassium permanganate, dissolve in 4mL pH 5 acetic acid buffer at room temperature;
2.称取5mg葡萄糖氧化酶，室温溶解于4ml pH 5的乙酸缓冲液中；2. Weigh 5mg glucose oxidase, dissolve in 4ml pH 5 acetate buffer at room temperature;
3.量取2mL步骤二的葡萄糖氧化酶溶液，加入40uL步骤一的高锰酸钾溶液，翻转混匀，取其中1mL用pH 5的乙酸缓冲液稀释至20mL，得到标准检测溶液；3. Measure 2mL of the glucose oxidase solution in step 2, add 40uL of potassium permanganate solution in step 1, invert and mix, take 1mL of it and dilute it to 20mL with acetic acid buffer of pH 5 to obtain a standard detection solution;
4.量取20uL步骤一的高锰酸钾溶液，用pH 5的乙酸缓冲液稀释至20mL，翻 转混匀，得到标准空白溶液。4. Measure 20uL potassium permanganate solution of step 1, dilute to 20mL with the acetic acid buffer of pH 5, invert and mix to obtain standard blank solution.
实施例3Example 3
特定类别下数据模型的建立，以BSA溶液样本为例。The establishment of the data model under a specific category, taking the BSA solution sample as an example.
1.称取2g牛血清白蛋白，室温溶解于40mL pH 5的乙酸缓冲溶液中，得到50g/L BSA溶液；1. Weigh 2g of bovine serum albumin, dissolve in 40mL acetic acid buffer solution of pH 5 at room temperature to obtain 50g/L BSA solution;
2.称取0.18g无水葡萄糖，室温溶解于10mL步骤一的BSA溶液中，得到葡萄糖浓度为100mM的BSA溶液；2. Weigh 0.18 g of anhydrous glucose, dissolve it in 10 mL of the BSA solution of step 1 at room temperature, and obtain a BSA solution with a glucose concentration of 100 mM;
3.分别量取40，80，120，160，200，240，280，320，360，400，440，480，520，560，600，640，680，720，760，800uL步骤二的葡萄糖浓度为100mM的BSA溶液，用步骤一的BSA溶液定容至4mL，得到葡萄糖浓度分别为1，2，3，4，5，6，7，8，9，10，11，12，13，14，15，16，17，18，19，20mM的BSA溶液，放置于4℃预冷；3. Measure 40, 80, 120, 160, 200, 240, 280, 320, 360, 400, 440, 480, 520, 560, 600, 640, 680, 720, 760, 800uL respectively. The glucose concentration in step 2 is 100mM BSA solution, dilute to 4mL with the BSA solution in step 1, and obtain glucose concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20mM BSA solution, pre-cooled at 4°C;
4.分别量取500uL步骤三得到的各浓度BSA溶液，加入1000uL同样在4℃预冷的无水乙醇，翻转混匀，放置于4℃沉淀20分钟，室温离心(3500转/分钟，10分钟)；4. Measure 500uL of each concentration of BSA solution obtained in step 3, add 1000uL of absolute ethanol that is also pre-cooled at 4°C, invert and mix, place at 4°C for precipitation for 20 minutes, and centrifuge at room temperature (3500 rpm for 10 minutes). );
5.将步骤四离心所得上清液，分A、B组各取150uL，置于干燥箱85℃蒸发至完全。5. The supernatant obtained by centrifugation in step 4 was divided into groups A and B to take 150uL each, and placed in a drying box at 85°C to evaporate to completeness.
6.对步骤五所得的每一葡萄糖浓度样本的干燥物A、B组，分别加入用于检测葡萄糖浓度区间1-20mM的标准检测溶液及标准空白溶液各1mL，振荡溶解混匀，静置1小时使其充分反应；6. Add 1 mL each of the standard detection solution and standard blank solution for detecting the glucose concentration range of 1-20 mM to the dry matter A and B of each glucose concentration sample obtained in step 5, shake to dissolve and mix, and let stand for 1 mL. hours to make it fully react;
7.将步骤六所得的反应完成后的样本各取200uL装入7.5mm核磁共振测试管，使用Bruker mq60低场核磁共振测量仪，测量脉冲为IR，扫描次数8次，采样点数21，进行纵向弛豫时间测量；7. Take 200uL of the samples after the reaction obtained in step 6 and put them into a 7.5mm nuclear magnetic resonance test tube. Use a Bruker mq60 low-field nuclear magnetic resonance measuring instrument. The measurement pulse is IR, the number of scans is 8 times, and the number of sampling points is 21. relaxation time measurement;
8.以葡萄糖浓度作为横坐标，每一葡萄糖浓度样本A、B组间的纵向弛豫时间之差作为纵坐标，绘制出BSA溶液样本葡萄糖含量检测的数据模型标准曲线，如附图2所示，记为与BSA检测对象及Bruker mq60低场核磁共振测量仪对应的数据模型。8. Take the glucose concentration as the abscissa, and the difference between the longitudinal relaxation times of each glucose concentration sample A and B group as the ordinate, draw the data model standard curve of the BSA solution sample glucose content detection, as shown in accompanying drawing 2 , denoted as the data model corresponding to the BSA detection object and the Bruker mq60 low-field NMR measuring instrument.
上述实施例为本发明针对牛血清白蛋白溶液样本的一种实施方式，但本发明的实施方式并不受上述实例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiment is an embodiment of the present invention for the bovine serum albumin solution sample, but the embodiment of the present invention is not limited by the above-mentioned example, and any other changes made without departing from the spirit and principle of the present invention, Modifications, substitutions, combinations, and simplifications should all be equivalent substitutions, which are all included within the protection scope of the present invention.
Claims (10)
- 一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，以葡萄糖氧化酶和酸化高锰酸钾作为反应底物，并利用有机试剂除去生物样本中原有的蛋白质干扰，通过 1H-NMR弛豫检测高锰酸钾弛豫时间的变化来计算被测样本中葡萄糖含量，该方法具体包括如下步骤： A relaxation nuclear magnetic resonance method for detecting glucose content in a liquid biological sample, which is characterized in that glucose oxidase and acidified potassium permanganate are used as reaction substrates, and organic reagents are used to remove the original protein interference in the biological sample. -NMR relaxation detects the change of potassium permanganate relaxation time to calculate the glucose content in the tested sample, and the method specifically includes the following steps:步骤a.将被测液体生物样本加入无水乙醇或其他具有相同效果的有机试剂混匀后静置，使样本中大蛋白变性沉淀：液体生物样本事先进行预冷，随后加入同样经预冷的无水乙醇或其他有机试剂，充分翻转混匀后在预冷温度下静置，等待发生变性的蛋白质充分沉淀；Step a. Add anhydrous ethanol or other organic reagents with the same effect to the liquid biological sample to be tested, mix well and let stand to denature and precipitate large proteins in the sample: the liquid biological sample is pre-cooled in advance, and then the same pre-cooled Absolute ethanol or other organic reagents, fully invert and mix, and let stand at pre-cooling temperature to wait for the denatured protein to fully precipitate;步骤b.取上层清液蒸发残留有机试剂：将产生沉淀的样本液进行离心，取出分离的上清液，分为等量的两份记为A、B组，置于干燥箱充分蒸发；Step b. Evaporate the residual organic reagent from the supernatant: centrifuge the precipitated sample solution, take out the separated supernatant, divide it into two equal parts and record them as groups A and B, and place them in a drying box to fully evaporate;步骤c.将A、B组分别加入标准检测溶液及对照组空白溶液进行反应：A组加入含有葡萄糖氧化酶及高锰酸钾的酸性溶液，空白对照组B组加入只含有等浓度高锰酸钾的酸性溶液，分别混匀；Step c. Add standard detection solution and control group blank solution to group A and B respectively to react: group A is added with an acidic solution containing glucose oxidase and potassium permanganate, and group B of blank control group is added with permanganic acid containing only equal concentration Potassium acid solution, mix separately;步骤d.用核磁共振仪器检测A、B组弛豫时间之差：将最终反应完成的A、B组分别取样进行核磁共振检测其弛豫时间T 1、T 2或其他可表征样本弛豫的测量量，两组弛豫时间的差值记为ΔT； Step d. Detect the difference between the relaxation times of groups A and B with a nuclear magnetic resonance instrument: respectively sample the groups A and B after the final reaction is completed, and perform nuclear magnetic resonance detection of their relaxation times T 1 , T 2 or other parameters that can characterize the relaxation of the sample. The measured quantity, the difference between the relaxation times of the two groups is recorded as ΔT;步骤e.根据测得的弛豫时间差与该样本种类已知的数据模型对照得出被测生物样本的葡萄糖含量：将被测液体生物样本的A、B组ΔT作为输入量代入已知的数据模型，得出的输出量即为测得的葡萄糖含量。Step e. Comparing the measured relaxation time difference with the known data model of the sample type to obtain the glucose content of the tested biological sample: Substitute the A and B groups ΔT of the tested liquid biological sample as the input into the known data model, and the resulting output is the measured glucose content.
- 根据权利要求1所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述被测液体生物样本包括全血、血清、尿液、唾液及含有葡萄糖且质地为液体的生物来源类样本，其中具有一些干扰检测的白蛋白、球蛋白、粘蛋白，在有机溶剂中发生变性，产生絮状或团块状沉淀物。A method of relaxation nuclear magnetic resonance for detecting glucose content in a liquid biological sample according to claim 1, wherein the liquid biological sample to be tested comprises whole blood, serum, urine, saliva, and glucose-containing and liquid texture Samples of biological origin, which have some albumin, globulin, and mucin that interfere with the detection, are denatured in organic solvents, resulting in flocculent or clump-like precipitates.
- 根据权利要求1所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述蒸发残留是将液体样本敞口置于干燥箱中直至其中有机溶剂在高于其沸点的环境中蒸发完全。A relaxation nuclear magnetic resonance method for detecting glucose content in a liquid biological sample according to claim 1, characterized in that, the evaporation residue is to place the liquid sample open in a drying box until the organic solvent is above its boiling point. evaporated completely in the environment.
- 根据权利要求1所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述标准检测溶液是将固体葡萄糖氧化酶及高锰酸钾溶解于pH 5的乙酸缓冲液中。A relaxation nuclear magnetic resonance method for detecting glucose content in a liquid biological sample according to claim 1, wherein the standard detection solution is to dissolve solid glucose oxidase and potassium permanganate in an acetic acid buffer of pH 5 middle.
- 根据权利要求4所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述的葡萄糖氧化酶，其含量根据被测生物样本中葡萄糖含量的量级不同而有所区别；所述的高锰酸钾，其含量根据被测生物样本所需的检测上限不同而有所区别。The relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample according to claim 4, wherein the content of the glucose oxidase varies according to the magnitude of the glucose content in the tested biological sample. Difference; the content of potassium permanganate varies according to the upper limit of detection required by the biological sample to be tested.
- 根据权利要求1所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述对照组空白溶液是将高锰酸钾溶解于pH 5的乙酸缓冲液中，终浓度与标准检测溶液中的高锰酸钾浓度一致。A relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample according to claim 1, wherein the blank solution of the control group is to dissolve potassium permanganate in an acetic acid buffer of pH 5, and the final concentration Consistent with the potassium permanganate concentration in the standard detection solution.
- 根据权利要求1所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述核磁共振仪器为满足 1H-NMR弛豫信号测量要求的低场核磁共振仪器，取样测量的样本量根据仪器要求有所不同。 The relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample according to claim 1, wherein the nuclear magnetic resonance instrument is a low-field nuclear magnetic resonance instrument that meets the measurement requirements of 1 H-NMR relaxation signals, and the sampling The measured sample size varies according to the instrument requirements.
- 根据权利要求1所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述弛豫时间利用反转回复脉冲序列IR或多回波测量序列CPMG测得。The relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample according to claim 1, wherein the relaxation time is measured by using an inversion recovery pulse sequence IR or a multi-echo measurement sequence CPMG.
- 根据权利要求1所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述已知的数据模型是对基础成分与被测样本一致，即同属血清、尿液或唾液，并且其葡萄糖含量已知的指定生物样本进行如上步骤的检测后获得的一系列ΔT与对应的葡萄糖浓度联立得到的数据模型，每类生物样本都具有与之对应的不同数据模型。A relaxation nuclear magnetic resonance method for detecting glucose content in a liquid biological sample according to claim 1, wherein the known data model is based on the same basic components as the tested sample, that is, both belong to serum, urine or A series of ΔT and corresponding glucose concentrations are obtained after the detection of saliva and a designated biological sample whose glucose content is known in the above steps are simultaneously obtained. Each type of biological sample has a different data model corresponding to it.
- 根据权利要求1所述的一种检测液体生物样本葡萄糖含量的弛豫核磁共振方法，其特征在于，所述被测样本的检测仪器需与建立数据模型使用的弛豫核磁共振检测仪器一致，具有相同的质子共振频率、测试温度，使用相同的测量脉冲序列、扫描次数、回波间隔的一系列测量参数。The relaxation nuclear magnetic resonance method for detecting the glucose content of a liquid biological sample according to claim 1, wherein the detection instrument of the tested sample must be consistent with the relaxation nuclear magnetic resonance detection instrument used for establishing the data model, and has The same proton resonance frequency, test temperature, and a series of measurement parameters using the same measurement pulse sequence, scan times, and echo interval.
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