阅读说明：本技术 一种用于测量钙离子的试剂盒及其使用方法 (Kit for measuring calcium ions and use method thereof ) 是由 柯成锋 于 2020-12-24 设计创作，主要内容包括：本发明属于分析检测技术领域,具体涉及一种用于测量钙离子的试剂盒及其使用方法。本发明主要涉及一种含有如下式I所示化合物的用于测量钙离子的试剂盒及其使用方法。所述试剂盒稳定性好、精密度高、线性范围广、不受镁干扰、且不含环境不友好的砷元素,可以应用于生化及临床医药领域。(The invention belongs to the technical field of analysis and detection, and particularly relates to a kit for measuring calcium ions and a using method thereof. The invention mainly relates to a kit containing a compound shown as the following formula I and used for measuring calcium ions and a using method thereof. The kit has good stability, high precision, wide linear range, no interference of magnesium and no environmentally unfriendly arsenic element, and can be applied to the fields of biochemistry and clinical medicine.)

1. A compound of the formula I, wherein,

wherein R is₁、R₄Identical or different, independently of one another, from hydrogen, halogen, carboxyl, C_1-6Alkyl and formyl;

R₂、R₃、R₅and R₆Identical or different, independently of one another, from hydrogen, halogen, C_1-6Alkyl or C_1-6An alkoxy group;

X⁺are positively charged counterions.

2. A compound of claim 1, wherein R is₁、R₄Identical or different, independently of one another, from hydrogen, halogen, carboxyl, C_1-3Alkyl and formyl;

R₂、R₃、R₅and R₆Identical or different, independently of one another, from hydrogen, halogen, C_1-3Alkyl or C_1-3An alkoxy group;

X⁺independently potassium, sodium, lithium or cesium ions.

3. A compound of claim 2, wherein R is₁、R₄Selected from hydrogen;

R₂is methyl or ethyl;

R₃、R₅and R₆Is H;

X⁺independently a potassium ion.

4. A compound according to claim 3, characterized in that the compound of formula I is selected from the group consisting of the following compounds NNM-BAPTA,

5. a kit for detecting the concentration of calcium ions, comprising: a compound of formula I according to any one of claims 1 to 4, a buffering agent and a releasing agent;

the buffer reagent enables the pH value of the detection system to be maintained at 7.0-11.0;

the releasing agent is selected from substances which have better performance in chelating calcium ions than the compound of the formula I.

6. The kit of claim 5, wherein the buffering agent is selected from a sodium barbiturate buffer system, a TRIS buffer system, a boric acid buffer system, a glycine buffer system, a carbonate buffer system, a phosphate buffer system, a CAPSO [ 3-cyclohexylamino) -2-hydroxy-1-propanesulfonic acid ] buffer system, a CAPS [ 3-cyclohexylamino) -2-propanesulfonic acid ] buffer system, or an imidazole buffer system;

preferably, the releasing agent is selected from EDTA, EGTA, DTPA, DTPMP, EDPMP, EDTA-2K, EDTA-2Na, EGTA-2K or EGTA-2 Na.

7. The kit of claim 5 or 6, wherein the releasing agent is at a concentration that is 5 times, 10 times, 20 times, or 50 times the concentration of NNM-BAPTA;

preferably, the concentration range of calcium ions detected by the kit is 0.0-5 mmol/L;

preferably, the kit for measuring calcium comprises a compound of formula I in a concentration of 0.05mmol/L to 50 mmol/L.

8. Use of a kit according to any one of claims 5-7 for the detection of calcium in a blood sample (e.g. whole blood, plasma or serum) or any other aqueous liquid sample (e.g. cerebrospinal fluid, lymph, saliva or urine).

9. A method for determining the concentration of calcium ions in a sample using a kit according to any one of claims 5 to 7, comprising the steps of: mixing the sample with a solution comprising a compound of formula I and a buffer, thereby binding calcium ions to the compound of formula I, then adding a release agent to release calcium ions from the binding of the compound of formula I with calcium ions, wherein the release results in a change in absorbance of the compound of formula I, measuring the change in absorbance and using the measured change in absorbance to determine the calcium ion concentration.

10. The method according to claim 9, wherein the sample is a blood sample (e.g. whole blood, plasma or serum) or any other aqueous liquid sample (e.g. cerebrospinal fluid, lymph, saliva or urine).

Preferably, the calcium ion concentration is in a direct proportion relation with the change of absorbance, and the calcium ion concentration in the sample of interest can be obtained according to standard operation, and is specifically calculated by using the following formula:

ca concentration (mmol/L) ═ a_{Measurement of}-A_{Blank space})/(A_{Calibration article}-A_{Blank space}))×C_{Calibration article}，

Wherein A is_{Measurement of}Is the measured value of the absorbance of the sample to be measured, A_{Blank space}Value of absorbance of blank sample, A_{Calibration article}The absorbance value of the calibrator.

Technical Field

The invention belongs to the technical field of analysis and detection, and particularly relates to a kit for measuring calcium ions and a using method thereof.

Background

The total calcium content in serum is a common clinical biochemical test index and has important clinical significance.

Calcium is the most abundant cation in the human body, the reference range is very narrow (2.08-2.70mmol/L), and the medically determined horizontal concentrations are 1.75mmol/L, 2.75mmol/L and 3.38mmol/L, respectively. Slight deviations above or below these levels are diagnostic for a number of physiological disorders. Serum calcium elevation is commonly seen in hyperparathyroidism, multiple myeloma, sarcoidosis, and intestinal excess absorption resulting from massive vitamin D therapy. Reduced serum calcium levels are typically associated with hypoparathyroidism, chronic renal failure, and the like.

The method for measuring the total calcium in the serum mainly comprises a flame photometry, an atomic absorption spectrophotometry, a radionuclide dilution mass spectrometry, a titration method and a colorimetric method, wherein the decisive method is the radionuclide dilution mass spectrometry, and the reference method is the atomic absorption spectrophotometry. Although the flame photometry, the atomic absorption spectrophotometry and the radionuclide dilution mass spectrometry have accurate results and few interference factors, the equipment is complex and expensive, and is not suitable for routine experiments and automatic analysis.

The commonly used methods for measuring calcium in clinical examinations are colorimetric methods, such as the o-cresolphthalein complex ketone (OCPC) method, the azo arsenic III method, the methyl thymol blue method, and the NM-BAPTA method. Although these methods are currently in use on the market, each method has drawbacks.

Although the OCPC method is a conventional method recommended by WHO and clinical laboratory center of Ministry of health of China (1997), the method has the serious defects of poor selectivity (magnesium can be combined), poor stability (excessively depending on strong alkaline environment), no zero-point-passing linearity and the like.

The azoarsenic III method is a method developed in recent years, eliminates many defects of the OCPC method, and has the advantages of stable reagent, low background absorbance, no strong base, no magnesium interference and the like, but has the problems of chemical pollution among reagents, low sensitivity, environmental pollution and the like.

The method for measuring serum calcium by methyl thymol blue colorimetry has the characteristics of rapidness, simplicity, convenience and accurate result, is suitable for clinical biochemical examination, and has the defect that the reagent is not ideal in stability due to strong alkalinity.

The NM-BAPTA process has the disadvantage of low yield of nitration of the developer. Moreover, the NM-BAPTA method is exclusively matched with relevant equipment sold by Roche for use, so the method has the defects of high price, few types of applicable equipment, inconvenience for clinical application and popularization and the like.

In conclusion, the development of a calcium detection reagent which has good stability, high precision, wide linear range, no interference from magnesium, no arsenic element, applicability to clinical and full-automatic detection and wide application range is an urgent problem to be solved at present.

Disclosure of Invention

The present invention aims to provide a novel calcium detection reagent to improve or eliminate the defects of the calcium detection reagents in the prior art.

The inventors have found that the bis-nitro substituted BAPTA-type chelating agents show very good properties in this respect, being very suitable for the detection of calcium ions.

The invention firstly provides a compound shown in the formula I,

wherein R is₁、R₄Identical or different, independently of one another, from hydrogen, halogen, carboxyl, C_1-6Alkyl and formyl;

R₂、R₃、R₅and R₆Identical or different, independently of one another, from hydrogen, halogen, C_1-6Alkyl or C_1-6An alkoxy group;

X⁺are positively charged counterions.

According to an embodiment of the invention, R₁、R₄Identical or different, independently of one another, from hydrogen, halogen, carboxyl, C_1-3Alkyl and formyl;

R₂、R₃、R₅and R₆Identical or different, independently of one another, from hydrogen, halogen, C_1-3Alkyl or C_1-3An alkoxy group;

X⁺independently potassium, sodium, lithium or cesium ions.

According to a preferred embodiment of the invention, R₁、R₄Selected from hydrogen;

R₂is methyl or ethyl;

R₃、R₅and R₆Is H;

X⁺independently a potassium ion.

By way of example, the compounds of formula I are selected from the following compounds NNM-BAPTA,

the invention also provides a kit for detecting the concentration of calcium ions, which comprises: compounds of formula I as described above, buffering agents and release agents;

the buffer reagent enables the pH value of the detection system to be maintained at 7.0-11.0;

the releasing agent is selected from substances which have better performance in chelating calcium ions than the compound of the formula I.

According to an embodiment of the invention, the buffer reagent maintains the pH of the detection system between 7.0 and 10.0.

According to an embodiment of the invention, the buffer reagent is selected from the group consisting of a barbiturate sodium buffer system, a TRIS buffer system, a boric acid buffer system, a glycine buffer system, a carbonate buffer system, a phosphate buffer system, a CAPSO [ 3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid ] buffer system, a CAPS [ 3-cyclohexylamino-2-propanesulfonic acid ] buffer system or an imidazole buffer system.

According to an embodiment of the invention, the releasing agent is selected from EDTA (ethylenediaminetetraacetic acid, CAS registry number 60-00-4), EDTA-2K (dipotassium ethylenediaminetetraacetic acid, CAS registry number 25102-12-9), EGTA (ethylene glycol-bis (2-aminoethyl ether) -N, N, N, N-tetraacetic acid, CAS registry number 67-42-5), DTPA (diethylenetriaminopentaacetic acid, CAS registry number 67-43-6), DTPMP (diethylenetriaminopenta (methylenephosphonic acid), CAS registry number 15827-60-8) and/or EDPMP (ethylenediaminetetra (methylenephosphonic acid), CAS registry number 1429-50-1).

According to embodiments of the invention, the concentration of the releasing agent is 5 times, 10 times, 20 times, or 50 times the concentration of the NNM-BAPTA, or any two of these values are within a range consisting of the upper and lower limits.

According to an embodiment of the invention, the kit detects calcium ion at a concentration ranging from 0.0 to 5mmol/L, such as 0.0001 to 3mmol/L, e.g. 0.2 to 1 mmol/L.

According to an embodiment of the invention, the final concentration of the compound of formula I in the kit will be at least 1.1 to 2.5 times, 3 times and at most 20 times, 15 times or 10 times the concentration of calcium ions in the test sample, calculated for a sample having an expected upper limit of 5 mmol/L. Preferably, the final concentration of the compound of formula I in the assay mixture will be at least 1.1 times, 2 times, 2.5 times, 3 times and at most 20 times, 15 times or 10 times the molar concentration obtained by multiplying the dilution factor of the sample by 5 mmol/L. For example, in the case where the sample is diluted 1:100, the final calcium ion concentration in the assay mixture will be 0.05 mmol/L. The final concentration of the compound of formula I should be at least 1.1 times this concentration, i.e. 0.055 mmol/L.

In a preferred embodiment, the kit for measuring calcium comprises a compound of formula I in a concentration of 0.05mmol/L to 50 mmol/L.

According to an embodiment of the present invention, the kit for detecting calcium ion concentration may comprise two or more compounds of formula I. In a preferred embodiment, the kit for detecting the concentration of calcium ions comprises a single compound of formula I.

According to an embodiment of the present invention, the kit for detecting calcium ion concentration may further comprise or not comprise a detergent. Preferably, the kit for detecting the calcium ion concentration does not contain a detergent. In the calcium ion detection process, a detergent is added to a reagent for measuring calcium ions to reduce interfering non-specific binding, reduce bubbles and air bubbles, and the like. Preferably, the detergent refers to an ionic detergent or a non-ionic detergent. Examples of detergents include, but are not limited to: sodium Dodecyl Sulfate (SDS), fatty acid salt,Family, octyl glucoside, 3- [ (3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate (CHAPS), sodium dodecyl-maltoside (DM), lauryl diethylamine oxide (LDAO), NP-40 and family, primary amines, amine acetates and amine hydrochlorides, quaternary ammonium salts, trimethylEthylammonium bromide, amides of substituted diamines, diethanolaminopropylamine or diethylaminopropionamide (diethylenediaminopropylamide), amides of cyclized diethylenetriamine, alkylarylsulfonates, petroleum sulfonates, sulfonated glycerol esters, cholamides (cholamides), sulfobetaines (sulfobetaines), alkylglycosides, saponins, alkyl-polyglycol ethers.

The present invention also provides the use of a kit for detecting calcium ion concentration as described above for detecting calcium in a blood sample (e.g. whole blood, plasma or serum) or any other aqueous liquid sample (e.g. cerebrospinal fluid, lymph, saliva or urine).

The invention also provides a method for determining the calcium ion concentration in a sample by using the kit for detecting the calcium ion concentration, which comprises the following steps: mixing the sample with a solution comprising a compound of formula I and a buffer, thereby binding calcium ions to the compound of formula I, then adding a release agent to release calcium ions from the binding of the compound of formula I with calcium ions, wherein the release results in a change in absorbance of the compound of formula I, measuring the change in absorbance and using the measured change in absorbance to determine the calcium ion concentration.

Thus, according to embodiments of the present invention, the sample may be treated without detergent prior to detection.

According to an embodiment of the invention, the sample is a blood sample (e.g. whole blood, plasma or serum) or any other aqueous liquid sample (e.g. cerebrospinal fluid, lymph, saliva or urine).

According to the embodiment of the present invention, the calcium ion concentration is in a direct proportion relation with the change of absorbance, and the calcium ion concentration in the sample of interest can be obtained according to standard operation, and is calculated by using the following formula:

ca concentration (mmol/L) ═ a_{Measurement of}-A_{Blank space})/(A_{Calibration article}-A_{Blank space}))×C_{Calibration article}。

Wherein A is_{Measurement of}Is the measured value of the absorbance of the sample to be measured, A_{Blank space}Value of absorbance of blank sample, A_{Calibration article}The absorbance value of the calibrator.

Compounds of formula I herein are capable of measuring calcium ion concentration by the following principle: first allowing the compound of formula I to bind calcium ions and then releasing the calcium ions by adding a releasing agent, thereby comparing the change in absorbance before and after the release of the calcium ions; since the change in absorbance is in direct proportion to the calcium ion concentration, the calcium ion concentration in the sample can be calculated from the change in absorbance.

The invention has the advantages of

The calcium detection reagent containing the compound shown in the formula I has good stability, high precision, wide linear range, no interference of magnesium and no environmentally-unfriendly arsenic element, can be applied to the fields of biochemical and clinical medicine, and does not use a detergent which is usually necessary in the field when being prepared into a kit.

Definition and description of terms

The term "halogen" refers to F, Cl, Br and I. In other words, F, Cl, Br, and I may be described as "halogen" in the present specification.

The term "C_1-6Alkyl "is understood to mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3, 4, 5 or 6 carbon atoms. The alkyl group is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-ethylpropyl group, a 1, 2-dimethylpropyl group, a neopentyl group, a 1, 1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a 3, 3-dimethylbutyl group, a 2, 2-dimethylbutyl group, a 1, 1-dimethylbutyl group, a 2, 3-dimethylbutyl group, a 1, 3-dimethylbutyl group or a 1, 2-dimethylbutyl group.

The term "C_1-6Alkoxy "means C_1-6alkyl-O-wherein C_1-6Alkyl groups have the definitions as described above.

Drawings

FIG. 1 is a scheme showing the synthesis of NNM-BAPTA in example 1.

FIG. 2 is a one-dimensional representation of the NNM-BAPTA synthesized in example 1¹H NMR spectrum (solvent D)₂O)。

FIG. 3 is a two-dimensional representation of the NNM-BAPTA synthesized in example 1¹H NMR spectrum (solvent D)₂O)。

FIG. 4 shows the results of measuring the calcium ion concentration on a Toshiba TBA-40FR full-automatic biochemical analyzer according to example 2. In the upper part (a), the theoretical values and the actual measured values are plotted against one another. In the lower part (B), the recovery values are given.

FIG. 5 is a graph showing the results of measuring the calcium ion concentration on a Toshiba TBA-40FR full-automatic biochemical analyzer according to example 2, wherein the theoretical value and the actual measurement value are plotted against each other.

FIG. 6 is the results of measuring the calcium ion concentration of NNM-BAPTA on a Roche C8000701 module fully automated biochemical analyzer, according to example 6, where theoretical and actual measurements are plotted against each other.

FIG. 7 is the result of measuring the calcium ion concentration by NM-BAPTA on a Roche C8000701 module fully automated biochemical analyzer, according to example 6, in which the theoretical value and the actual measured value are plotted against each other.

FIG. 8 shows the absorption spectra read after analysis "sample + R1" and after analysis "sample + R1+ R2", respectively, according to example 2. The indices of the two spectrograms are given below the graph.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise specified, the reagents used in the following examples are all commercially available products or can be prepared by known methods.

Example 1

The synthetic route of the NNM-BAPTA is shown in figure 1, and specifically comprises the following steps:

(1) synthesis of 2- (2-chloroethoxy) nitrobenzene

300g of 2-nitrophenol and 620g of 1-bromo-2-chloroethane were dissolved in 800mL of Dimethylformamide (DMF), and after stirring well, 300g of potassium carbonate was added thereto, heated under reflux and stirred overnight. After cooling to room temperature, extraction was carried out 3 times with ethyl acetate, and after the organic phase was dried by spinning, recrystallization was carried out with methanol to obtain 300g of the objective product.

(2) Synthesis of 2- (2- (2-nitrophenoxy) ethoxy) -4-methylnitrobenzene

300g of 2- (2-chloroethoxy) nitrobenzene and 230g of 2-nitro-5-methylphenol were dissolved in 1000ml of DMF, and after stirring well, 220g of potassium carbonate was added thereto, heated under reflux and stirred overnight. After cooling to room temperature, the reaction solution was poured into cold water, and the solid was collected and recrystallized from methanol to obtain 400g of the objective product.

(3) Synthesis of 2- (2- (2-aminophenoxy) ethoxy) -4-methylaniline (Compound a)

250g of 2- (2- (2-nitrophenoxy) ethoxy) -4-methylnitrobenzene were dissolved in 1800mL of D MF, 25g of 20% Pd/C was added thereto, and the mixture was stirred under 10psi of hydrogen overnight with heating. After the reaction, Pd/C is filtered out, and the filtrate is dried by spinning to obtain 200g of solid product.

(4) Synthesis of N- (2- (2- (di (methoxycarbonylmethyl) amino) -phenoxy) -ethoxy) -4-methylphenyl), N-methoxycarbonylmethyl-amino-acetic acid methyl ester (Compound b)

200g of 2- (2- (2-aminophenoxy) ethoxy) -4-methylaniline (compound a) and 600g of bromo-acetic acid methyl ester were dissolved in 1000mL of acetonitrile, and after stirring well, 500g of potassium carbonate was added thereto. Heat to reflux and stir overnight. After cooling to room temperature, the solid was removed by filtration, and the filtrate was dried by spinning and then recrystallized from methanol to obtain 215g of the objective product.

(5) Synthesis of N- (2- (2- (bis (methoxycarbonylmethyl) amino) -4-nitrophenoxy) -ethoxy) -4-methyl-5-nitrophenyl), N-methoxycarbonylmethyl-amino-acetic acid methyl ester (Compound c)

100g of ((2- (2- (2- (di-methoxycarbonylmethyl-amino) -phenoxy) -ethoxy) -4-methylphenyl) -methoxycarbonylmethyl-amino) -acetic acid methyl ester (compound b) were dissolved in 1000ml of concentrated sulfuric acid. After stirring uniformly at 0 ℃ and then adding 10mL of nitric acid thereto, the reaction was carried out at room temperature for 2 hours. The reaction solution was poured directly into an ice-water mixture to precipitate a solid. The solid was collected by filtration, washed with water 3 times and then dried, and then recrystallized from methanol to obtain 88g of the objective product.

(6) Synthesis of N- (2- (2- (bis (methoxycarbonylmethyl) amino) -4-nitrophenoxy) -ethoxy) -4-methyl-5-nitrophenyl), N-methoxycarbonylmethyl-aminoacetic acid (Compound d)

20g of ((2- (2- (2- (di-methoxycarbonylmethyl-amino) -4-nitrophenoxy) -ethoxy) -4-methyl-5-nitrophenyl) -methoxycarbonylmethyl-amino) -acetic acid methyl ester (compound c) were dissolved in 150mL of methanol, followed by addition of 50mL of 4mol/L potassium hydroxide. Heating reflux reaction for 2 hours, cooling to room temperature, and rotary evaporation to remove methanol. Then 150mL of water was added to adjust the pH to acidity. Extract 3 times with ethyl acetate and combine the organic phases. The organic solvent was removed by rotary evaporation to obtain 12g of the target product.

(7) Synthesis of Potassium salt of Compound d

12g of compound d was dissolved in methanol and reacted by adding potassium hydroxide/methanol in an equimolar amount to compound d. After evaporation and drying, 15g of the expected product are obtained. One and two dimensional nuclear magnetic hydrogen spectra data are shown in figures 2 and 3.

Example 2

Measurement of calcium ion Using NNM-BAPTA prepared in example 1

An aliquot of the sample of interest was mixed well with a buffer solution containing compound NNM-BAPTA and incubated at 37 ℃ for 5 minutes. On different brands of fully automatic analyzers, this reagent is called R1.

The absorbance values at the optimum wavelength for the analytical sample and the R1 mixed solution were then read.

EDTA-2K was then added to the sample and R1 mixed solution, mixed well and incubated at 37 ℃ for 5 minutes. On different brands of fully automatic analyzers, this reagent is called R2.

The absorbance values at the optimum wavelength for the analytical sample, R1 and R2, respectively, were then read.

The change of the absorbance is in direct proportion to the calcium ion concentration in the sample of interest, and the calcium ion concentration in the sample of interest can be obtained according to standard operation. The specific calculation formula is as follows:

ca concentration (mmol/L) ═ a_{Measurement of}-A_{Blank space})/(A_{Calibration article}-A_{Blank space}))×C_{Calibration article}

Wherein A is_{Measurement of}Is the measured value of the absorbance of the sample to be measured, A_{Blank space}Value of absorbance of blank sample, A_{Calibration article}The absorbance value of the calibrator.

In table 1, an overview of the recommended methods for measuring calcium ions on different brands of fully automatic analyzers is given.

Table 1: recommended pipetting volume (μ L) for measuring calcium ions

Table 2: respective compositions of R1 and R2

	Buffers or releasing agents	pH	NNM-BAPTA
				R1	CAPS(50mmol/L)	10.0	0.275mmol/L
R2	EDTA-2K(5.5mmol/L)	7.0	-

Example 3

Accuracy of calcium measurements using NNM-BAPTA

The compounds of formula I for measuring calcium ions according to the invention and the methods of use thereof have technical advantages, for example it shows very good accuracy. Recovery was tested by adding an internal standard to the serum sample. This will become apparent if the theoretically expected values are compared with the actually measured values, and specific examples are as follows.

Different calcium ion concentrations (i.e., theoretical values) were compared to actual measurements.

An illustrative representative example is given as FIG. 4, measured on a fully automated analyzer of Toshiba TBA-40 FR. The figure confirms the accuracy of the measurement. It is evident from figure 4 that the recovery achieved with NNM-BAPTA is between 97% and 103.5%, which reflects that this reagent is quite accurate for testing calcium ions over the whole concentration range studied.

Example 4

The linear dependence of calcium measurements using NNM-BAPTA shows technical superiority of the method for measuring calcium ions according to the invention, in particular it shows very good linear dependence in the usual calcium ion measurement range (0-5.0 mmol/L). This becomes obvious if the theoretically expected values are compared with the actually measured values.

In the method according to the invention, different calcium ion concentrations (theoretical values) are compared with the actual measured values.

Yet another representative example is given as FIG. 5, the values of which are measured on a fully automated analyzer of Toshiba TBA-40 FR. This figure demonstrates the wide/linear range of measurements made according to the invention, i.e. with outstanding technical quality properties. As can be seen from FIG. 5, the linear r value is ≧ 0.9998, which reflects reasonably good linearity over the entire concentration range studied.

Example 5

Accelerated stability review

To study the stability of NNM-BAPTA, NNM-BAPTA was stored at different pH environments. The storage ambient temperature was 37 ℃. Under otherwise comparable conditions, the stability of agents containing NNM-BAP TA in different pH environments was accelerated. Starting on day 0 (i.e. the day on which the accelerated stability test of the agent at 37 ℃ C. was started), the measured values of the agent after storage at 37 ℃ C were compared with the values measured for the agent not stored at 37 ℃.

The buffer systems used were respectively: 40mmol/L barbital sodium (pH 7.2), 50mmol/L boric acid (pH 8.0), 50mmol/L glycine (pH 9.8), 50mmol/L CAPSO (pH 10), 50mmol/L imidazole (pH 10), 50mmol/L CAPS (pH 10). The experimental data obtained are summarized in tables 3 to 8.

Table 3: stability of NNM-BAPTA at 40mmol/L barbiturate sodium (pH 7.2) at 37 deg.C

Table 4: stability of NNM-BAPTA at 50mmol/L boric acid (pH 8.0) at 37 deg.C

Table 5: stability of NNM-BAPTA at 50mmol/L Glycine (pH 9.8) at 37 ℃

Table 6: stability of NNM-BAPTA at 50mmol/L CAPSO (pH 10) at 37 deg.C

Table 7: stability of NNM-BAPTA at 50mmol/L imidazole (pH 10) at 37 ℃

Table 8: stability of NNM-BAPTA at 50mmol/L CAPS (pH 10) at 37 ℃

As can be seen from the data in tables 3-8, 1) the NNM-BAPTA containing reagent recovered calcium from 102% to 105% at pH 9.8 and pH 10.0, indicating that the reagent was stable at pH 10.0. 2) The reagent containing NNM-BAPTA has calcium ion recovered at 105% -110% under the environment of pH 7.2 and pH 8.0, which shows that the reagent is stable under the environment of pH 7.2 and pH 8.0. But stability is relatively better in a pH 10.0 environment.

Example 6

Linear Performance comparison test with the commercially available reagent for calcium ion testing NM-BAPTA (Roche, batch No. 27022701, attorney: Roche diagnostics, Ltd.)

Commercial NM-BAPTA method reagents were purchased and comparative tests were performed to examine the linearity index.

Dilution to high and low concentrations with calcium single element solution standard (gbw (e)080118 lot No. 16123): 0.2mmol/L and 5 mmol/L. Then mixing the high and low concentrations to at least 6 diluted concentrations (x)_i). The samples at each dilution concentration were tested in parallel 3 times using the kit, and the mean value (y) was determined_i). In diluted concentration (x)_i) As independent variable, to determine the mean value (y)_i) Linear regression equations and linear correlation coefficients r are solved for the dependent variables.

Various experimental values were measured on a fully automated analyzer in the roche C8000701 module. From the linear graph fitted in fig. 6, the linear equation y of NNM-BAPTA method reagent is 0.9937x +0.0487, R²0.9999, r 0.9999; from the fitted linear graph of fig. 7, the linear equation y of the NM-BAPTA method reagent is 0.9624x +0.0839, R²0.9985 and 0.9992. Wherein the r value of the NNM-BAPTA method reagent fitting curve is closer to 1.000, which shows that the measured value of the NNM-BAPTA method reagent is closer to the actual value in the offline range and has good linearity compared with the NM-BAPTA method.

Example 7

Precision performance comparison test with commercial NM-BAPTA

For diagnostic reagents, the precision index of the reagent is also crucial, therefore, the commercial NM-BAPTA method reagent is purchased, a comparison test is carried out, and the precision index is mainly considered.

The calcium single element solution standard (GBW (E)080261 lot 16012, designation value 2.5mmol/L) and 3 serum samples at different concentrations were tested using the NNM-BAPTA containing kit and the commercially available NM-BAPTA method reagents, respectively. Under the condition of repetition, the above samples were respectively tested and repeated 20 times, and the average value of the measured values was calculatedAnd Standard Deviation (SD), Coefficient of Variation (CV) is calculated. The values were measured on a fully automated analyzer in the roche C8000701 module. Comparative experimental data are summarized in table 9.

TABLE 9 precision comparison test data of NNM-BAPTA and commercial NM-BAPTA

From the experimental results in Table 9, the NNM-BAPTA method reagents according to the invention, whether testing standards or testing different levels of serum samples, are significantly better accurate than the commercial NM-BAPTA method reagents.

Example 8

Determination of magnesium ion interference using NNM-BAPTA

Mg was prepared in different concentrations according to interference Experimental guidelines (WS/T416-2013), page 15, 10 times the concentration of the recommended laboratory concentration in Table C.1 in appendix C²⁺And (4) standard solution. Mixing different concentrations of Mg²⁺Addition of standard solution to matrix serum, Mg²⁺The standard solution is added in an amount of 10% of the serum volume of the matrix, the control group is added with the same amount of water as a control, and Mg addition is detected²⁺Serum base of standard solution (C1) and no Mg addition²⁺The standard solution-based matrix serum (C2) was calculated according to the following formulas (1), (2) and (3):

interference concentration-measured with a sample of standard liquid (test sample) C1-measured without a sample of standard liquid (base sample) C2 … … … … … … … … … … … … … … … … … … … … … (2)

Relative interference concentration/substrate serum concentration x 100% … … … … … … … … … (3)

The experimental data are summarized in Table 10.

TABLE 10 Mg²⁺Interference concentration test results

Table 10 results show that Mg in the samples²⁺When the concentration reaches 2mmol/L, the deviation of the result of the NNM-BAPTA method reagent for detecting the calcium ion concentration is only 1.0 percent even if Mg²⁺When the concentration reached 5mmol/L, the deviation was only 3.16%. The normal concentration range of magnesium ions in human bodies is 0.75 mmol/L-1.02 mmol/L, so that the NNM-BAPTA method reagent is not interfered by magnesium ions when being clinically used for calcium ion measurement.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.