WO2023010613A1 - Relaxation nuclear magnetic resonance method for quantitatively measruing specific component in liquid biological sample - Google Patents

Abstract

A relaxation nuclear magnetic resonance (NMR) method for quantitatively measuring the content of a specific component in a liquid biological sample, the specific process comprising: step a): generating hydrogen peroxide related to the content of a trace target component in a liquid biological sample by means of specific one-step or several-step enzyme decomposition reaction; step b): the product hydrogen peroxide highly-sensitively oxidizing ferrous ions under the impact of the composition and causing the ¹H-NMR relaxation time measured and obtained by a sample solution to decrease; and step c): comparing same with a standard tube sample of a known concentration, and calculating the content of the target component in the detected sample. In the method, the content of various components in a liquid biological sample may be detected with high sensitivity, high specificity and high interference resistance, helping to further expand the application of NMR technology in biological detection.

Description

A Relaxation NMR Method for the Quantitative Detection of Specific Components in Liquid Biological Samples technical field

The invention belongs to the technical field of analysis and detection of biochemical methods, and in particular relates to a relaxation nuclear magnetic resonance method for quantitatively detecting a specific component in a liquid biological sample, and a composition and a reaction solution for improving detection performance.

Background technique

Relaxation nuclear magnetic resonance technology has great application prospects in the fields of food science and biological detection because of its fast and easy operation. However, due to the low resolution of relaxation nuclear magnetic resonance, it is difficult to distinguish multiple Therefore, the quantitative detection of small molecules in complex samples still has great limitations. Especially when applied to the detection of trace components in biological samples, it is easily affected by different levels of interfering substances in the sample matrix, resulting in detection errors or failure to detect. Therefore, the specificity, sensitivity, and Anti-interference has high requirements.

One relaxation NMR detection method known in the art is the paramagnetic ion-mediated magnetic sensor, which amplifies the weak signal of the target component into a liquid through a target-induced Fe ²⁺ /Fe ³⁺ relaxation switch The change in relaxation of the sample as a whole. However, their sensitivity is still relatively low, which cannot meet the application requirements of trace component detection. At the same time, in the actual clinical application, the oxidation-reduction process of paramagnetic ions is easily interfered by levodopa, uric acid, vit-C, bilirubin, glutathione and other substances, resulting in false measurement results, seriously affecting the accuracy of the measurement sex. In terms of improving the anti-interference performance of the relaxation NMR method, a solution proposed in the field is to pretreat biological samples by physical and chemical means to remove the interfering proteins as much as possible, but in practical applications, the samples Some small molecular compounds with redox properties cannot be simply eliminated, and redundant interfering components are easily introduced. In terms of improving the sensitivity of the relaxation NMR method, a solution proposed in the art is to use the chelate of potassium thiocyanate and iron ions to enhance the contrast of the relaxation signal, but the formed complex is insoluble in water , It is easy to cause uneven sampling during detection and affect the measurement results. At the same time, potassium thiocyanate has certain toxicity and is harmful to the environment.

In the field of relaxation nuclear magnetic resonance detection, there is still a lack of a simple, safe and effective solution that can simultaneously improve multiple aspects of detection performance in the quantitative detection of liquid biological samples, including but not limited to glucose, cholesterol, glycerol The trace detection sensitivity, anti-interference ability and detection specificity of triester.

Contents of the invention

Technical problem: The purpose of this 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 quantitative detection of the content of specific components in liquid biological samples, and involves a series of methods using The composition and the prepared reaction solution for improving the sensitivity, specificity and anti-interference of relaxation detection, more specifically, convert the target component into the quantitative detection of the product hydrogen peroxide through the enzymatic reaction method, and pass the The composition enhances the ¹ H-NMR relaxation signal difference caused by a trace amount of hydrogen peroxide, detects the change in the relaxation time of the reaction solution and compares the result with the standard tube to calculate the content of the target component.

Technical solution: a relaxation nuclear magnetic resonance method for quantitatively detecting the content of specific components in liquid biological samples, the specific steps are:

a) Through a specific enzymatic decomposition reaction, the trace specific component in the liquid biological sample, that is, the target component, generates a hydrogen peroxide product related to its content; the liquid biological sample is a sample derived from an organism and has a liquid texture ; The buffer in the specific enzymatic decomposition reaction is added with a composition to improve the detection performance, and the composition to improve the detection performance is composed of a specific type of enzyme, sorbitol, and ferrous salt;

b) The hydrogen peroxide product oxidizes ferrous ions with high sensitivity under the influence of the composition and causes a decrease in the ¹ H-NMR relaxation time measured in the sample solution;

c) Comparing the test result with the standard tube sample of known concentration and calculating the content of the target component in the tested sample.

The liquid biological samples include whole blood, serum, urine, saliva, tissue fluid.

The hydrogen peroxide products related to the generation of specific trace components refer to glucose and glucose oxidase, cholesterol ester and cholesterol esterase and cholesterol oxidase, triglyceride and lipase, glycerol kinase and phosphoglycerol oxidase. class combination.

The relaxation NMR method refers to rapidly detecting information of a sample at least including transverse or longitudinal relaxation time based on ¹ H-NMR time-domain signals.

The standard tube sample of known concentration refers to a standard solution corresponding to the current target component, and the concentration of the standard is known.

The specific type of enzyme is selected according to the current target component, including any one of glucose oxidase, cholesterol oxidase, and glycerol phosphate oxidase.

Described ferrous salt comprises any one in ferrous chloride, ammonium ferrous sulfate.

The final concentration of the specific type of enzyme is sufficient to specifically hydrolyze or oxidize the target component, the reaction process is accompanied by the product hydrogen peroxide, and the content of the final hydrogen peroxide product is proportional to the target component.

The final concentration of ferrous ions produced by the ferrous salt is optimally prepared according to the content range of the target components, so that the ferrous ions in the final solution will not be completely oxidized.

Beneficial effect:

The present invention provides a method for rapidly detecting the content of specific components in liquid biological samples by nuclear magnetic resonance relaxation method. Compared with the existing relaxation nuclear magnetic resonance technology for detecting liquid samples, the composition of the present invention is The biochemical reaction between the components can improve the detection sensitivity and specificity of the relaxation nuclear magnetic resonance method for the trace components in the sample, and convert the content of the target component into the content of the product hydrogen peroxide, and then into the relaxation value measured by NMR. The change of Yu time greatly improves the contrast of detection difference. The NMR relaxation method adopted by the invention has high efficiency and low detection limit, and the related composition can improve the detection performance, and is more suitable for the detection of trace components in biological samples.

Description of drawings

Fig. 1 is the specific application of the reaction solution of the present invention and the composition therein in the quantitative detection of glucose in blood.

Fig. 2 is the transverse relaxation curve and the corresponding relaxation time T ₂ of the solution in the measurement tube of Example 1 of the present invention.

Fig. 3 is the contrast of the result of measuring serum glucose content (embodiment 1) and the standard value (biochemical analyzer) by the method of the present invention, and its accuracy is better than commercially available common electronic blood glucose meters.

Detailed ways

The present invention will be further described below in conjunction with 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.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

A relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample, a composition and a reaction solution for improving detection performance of the present invention are realized through the following technical scheme:

In the first aspect, a relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample, the specific steps are:

a) Make the trace target components in the liquid biological sample generate hydrogen peroxide related to its content through specific one or several steps of enzymatic decomposition reaction;

b) The product hydrogen peroxide oxidizes ferrous ions with high sensitivity under the influence of the composition and causes a decrease in the ¹ H-NMR relaxation time measured in the sample solution;

c) Comparing the test result with the standard tube sample of known concentration and calculating the content of the target component in the tested sample.

in:

The liquid biological samples include whole blood, serum, urine, saliva, interstitial fluid and other samples derived from living organisms and having a liquid texture.

Described specific component refers to the component that can specifically generate hydrogen peroxide product through one or several steps of enzymatic decomposition reaction, can be glucose (glucose oxidase), cholesterol ester (cholesteryl esterase, cholesterol esterase, cholesterol oxidase), triglycerides (lipase, glycerol kinase, phosphoglycerol oxidase), etc.

The relaxation NMR method refers to rapid detection of transverse or longitudinal relaxation time of a sample based on ¹ H-NMR of time-domain signals.

The standard tube sample of known concentration refers to a standard solution corresponding to the current target component, and the concentration of the standard is known.

In a second aspect, the present invention provides the use of a composition in improving the detection performance of relaxation nuclear magnetic resonance. , the improvement refers to the improvement of sensitivity, specificity, and/or anti-interference performance.

In the definition of the present invention, the term "detection performance" mainly refers to sensitivity, specificity and anti-interference.

in:

The specific type of enzyme is selected according to the current target component, including but not limited to glucose oxidase, cholesterol oxidase, phosphoglycerol oxidase and the like.

The ferrous salt can be dissolved in a buffer to generate ferrous ions, including but not limited to ferrous chloride, ammonium ferrous sulfate and the like.

The purposes of composition among the present invention are embodied by following biochemical reaction respectively:

In a specific embodiment, cholesterol ester┄cholesterol ^esterase →cholesterol+fatty acid, cholesterol┄cholesterol ^oxidase →Δ ⁴ -cholestenone+H ₂ O ₂ , said particular kind of enzyme highly specifically The detection of cholesteryl esters and cholesterol is converted to the detection of hydrogen peroxide.

In a specific embodiment, glucose┄glucose ^oxidase →gluconic acid+H ₂ O ₂ , said specific class of enzymes converts the detection of glucose into the detection of hydrogen peroxide with high specificity.

In a specific embodiment, triglyceride ┄ ^lipase → glycerol + fatty acid, glycerol ┄ ^{glycerol kinase} → glycerol-3-phosphate, glycerol-3-phosphate ┄ ^{glycerol phosphate oxidase} → dihydroxyacetone phosphate + H ₂ O ₂ , said specific class of enzymes converts the detection of triglycerides to the detection of hydrogen peroxide with high specificity.

Ferrous ions + H ₂ O ₂ → ferric ions, the ferrous salt converts changes in hydrogen peroxide content into samples with high sensitivity through the ferromagnetic properties of ferrous ions/ferric ions The difference in relaxation time.

Sorbitol + H ₂ O ₂ → peroxyl free radicals, the peroxyl free radicals provided by the chain reaction of sorbitol and hydrogen peroxide can multiply the oxidation of ferrous ions, greatly improving the reaction solution to hydrogen peroxide sensitivity.

In the third aspect, the present invention provides a reaction solution for relaxation nuclear magnetic resonance detection, which is used for a series of high-sensitivity specific reactions with the target components in the detection sample, which contains the above-mentioned composition and a conventional buffer, Further samples are included.

in:

The final concentration of the specific type of enzyme is sufficient to cause specific hydrolysis or oxidation of the latter, the reaction process is accompanied by product hydrogen peroxide, and the content of the final hydrogen peroxide is proportional to the target component.

The final concentration of ferrous ions produced by the ferrous salt is prepared according to the content range of the target components, so that the ferrous ions in the final solution will not be completely oxidized.

The relaxation nuclear magnetic resonance method of the present invention quantitatively detects a specific component in a liquid biological sample. Compared with the existing relaxation nuclear magnetic resonance technology for measuring liquid samples, by introducing the composition of the present invention, the trace nuclear magnetic signal of the specific component The difference is converted into a more significant relaxation signal difference caused by the oxidation of metal ions, which can detect the content of various components in liquid biological samples with high sensitivity, high specificity, and high anti-interference. In particular, in the case of complex clinical tests, by adjusting different specific types of enzymes and corresponding reaction solutions, the method can be applied to the rapid detection of multiple indicators.

Example 1

The glucose content in human serum samples was detected (detection range 3-20mmol/L).

1. Prepare a reaction solution suitable for quantitative detection of glucose: pH 6.7 phosphate buffer 50mmol/L, glucose oxidase ≥ 3ng/mL, sorbitol 200mmol/L, ferrous chloride 0.5mmol/L;

2. Prepare 2 standard tube sample solutions: the concentration of glucose is 3mmol/L and 20mmol/L respectively, bovine serum albumin is 0.2%;

3. Take 4 test tubes and operate according to the table:

After mixing, incubate in a 37°C water bath for 15 minutes.

4. Measure the transverse relaxation time T ₂ of the solution in each test tube, and calculate whole blood glucose (mmol/L)=3+17*(standard tube 1-measurement tube)/(standard tube 1-standard tube 2).

Embodiment result is as follows (italics are calculated data):

the	Standard tube 1	Standard tube 2	Assay tube 1	Assay tube 2	Assay tube 3	Assay tube 4
T 2 /ms	2059.75	218.43	1757.53	1293.44	1595.93	1866.5
Glucose/mM	3	20	5.81	10.07	7.31	4.79

Example 2

The total cholesterol content in human serum samples was detected (detection range 2-10mmol/L).

1. Preparation of reaction solution suitable for quantitative detection of total cholesterol: pH6.7 phosphate buffer 50mmol/L, cholesterol esterase ≥ 0.4kU/L, cholesterol oxidase ≥ 0.2kU/L, sodium cholate 0.1%, sorbic acid Alcohol 50mmol/L, ferrous ammonium sulfate 0.5mmol/L;

2. Prepare 2 standard tube sample solutions: the concentrations of cholesterol standards are 2mmol/L and 10mmol/L respectively, and bovine serum albumin is 0.2%;

3. Take 4 test tubes and operate according to the table:

After mixing, incubate in a 37°C water bath for 15 minutes.

4. Measure the longitudinal relaxation time T ₁ of the solution in each test tube, and calculate serum total cholesterol (mmol/L)=2+8*(standard tube 1-measurement tube)/(standard tube 1-standard tube 2).

Example 3

The triglyceride content in human serum samples was detected (detection range 1-5 mmol/L).

1. Prepare a reaction solution suitable for the quantitative detection of triglycerides: pH 6.7 phosphate buffer 50mmol/L, lipase ≥ 3U/mL, glycerol kinase ≥ 0.2U/mL, glycerol phosphate oxidase ≥ 2.5U/mL , ATP≥0.15mmol/L, sodium cholate 0.1%, sorbitol 20mmol/L, ferrous ammonium sulfate 0.5mmol/L;

2. Prepare 2 standard tube sample solutions: the concentrations of triglyceride standards are 1mmol/L and 5mmol/L respectively, and bovine serum albumin is 0.2%;

3. Take 4 test tubes and operate according to the table:

After mixing, incubate in a 37°C water bath for 15 minutes.

4. Measure the longitudinal relaxation time T ₁ of the solution in each test tube, and calculate serum triglyceride (mmol/L)=1+4*(standard tube 1-measurement tube)/(standard tube 1-standard tube 2).

The above-mentioned examples are several implementations of the present invention for the quantitative detection of common biochemical indicators in serum, but the implementation of the present invention is not limited by the above-mentioned examples, and any other methods that do not deviate from the spirit and principles of the present invention Changes, modifications, substitutions, combinations, and simplifications should all be equivalent replacements, and are all included within the protection scope of the present invention.

Claims (9)

1. A relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample, characterized in that the specific steps are:

   a) Through a specific enzymatic decomposition reaction, the trace specific component in the liquid biological sample, that is, the target component, generates a hydrogen peroxide product related to its content; the liquid biological sample is a sample derived from an organism and has a liquid texture ; The buffer in the specific enzymatic decomposition reaction is added with a composition to improve the detection performance, and the composition to improve the detection performance is composed of a specific type of enzyme, sorbitol, and ferrous salt;

   b) The hydrogen peroxide product oxidizes ferrous ions with high sensitivity under the influence of the composition and causes a decrease in the ¹ H-NMR relaxation time measured in the sample solution;

   c) comparing with the test result of the standard tube sample of known concentration and calculating the content of the target component in the tested sample.
2. A relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample according to claim 1, wherein the liquid biological sample includes whole blood, serum, urine, saliva, and interstitial fluid.
3. A relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample according to claim 1, wherein said trace specific component generates a hydrogen peroxide product related to its content refers to Glucose and glucose oxidase, cholesterol ester and cholesterol esterase and cholesterol oxidase, triglyceride and lipase, glycerol kinase and phosphoglycerol oxidase such combinations.
4. A relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample according to claim 1, wherein the relaxation nuclear magnetic resonance method refers to a fast method based on ¹ H-NMR time domain signals. The detection sample at least includes information including the transverse or longitudinal relaxation time.
5. A relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample according to claim 1, wherein the standard tube sample of known concentration refers to the standard corresponding to the current target component solution, the concentration of the standard is known.
6. A relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample according to claim 1, wherein the specific type of enzyme is selected according to the current target component, including glucose oxidase, cholesterol Any of oxidase and glycerol phosphate oxidase.
7. A kind of relaxation nuclear magnetic resonance method for quantitative detection of specific component content in liquid biological sample according to claim 1, it is characterized in that, described ferrous salt comprises any in ferrous chloride, ferrous ammonium sulfate A sort of.
8. A relaxation nuclear magnetic resonance method for quantitatively detecting the content of a specific component in a liquid biological sample according to claim 1, wherein the final concentration of the specific type of enzyme is sufficient to specifically hydrolyze the target component or Oxidation, the reaction process is accompanied by the product hydrogen peroxide, and the content of the final hydrogen peroxide product is proportional to the target component.
9. A kind of relaxation nuclear magnetic resonance method for quantitative detection of specific component content in liquid biological sample according to claim 1, it is characterized in that, the final concentration of the ferrous ion that described ferrous salt produces is according to target component Optimizing the deployment of the content range, so that the ferrous ions in the final solution will not be completely oxidized.

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