CN100489503C - Method for measuring plasma calcium by triplex pyridine ruthenium electrochemical luminescence - Google Patents

Abstract

The present invention relates to a method for determining plasma calcium by using terpyridyl ruthenium electrochemical luminescence, belonging to the field of plasma calcium determination technology. The technical scheme adopted by said invention includes the following steps: firstly, adding excessive oxalic acid solution (concentration is greater than 5 mM/L) into 50-100 microliters of plasma, making oxalic acid and blood calcium be fully reacted, then utilizing centrifugal machine to make centrifugal separation to make calcium oxalate and large molecules in the blood be precipitated downwards to bottom portion, using supernatant containing oxalic acid and terpyridyl ruthenium solution whose pH is 5-7.5 and making them fully be mixed, then making electrochemical luminescence determination so as to obtain plasma calcium content.

Description

Method for Determination of Plasma Calcium by Electrochemiluminescence of Ruthenium Tripyridine

technical field

The invention relates to a method for measuring plasma calcium by ruthenium terpyridine electrochemiluminescence, and belongs to the field of plasma calcium determination.

Background technique

Calcium mainly exists in the bones and teeth of the human body, and the content in the extracellular fluid is very small, but it plays an important role in maintaining normal neuromuscular irritability, glandular secretion, and the activity of some enzyme systems, especially in the process of blood coagulation. Blood calcium concentration is regulated between bones, kidneys, and intestines, and at the same time, parathyroid hormone (increasing blood calcium), calcitonin (lowering blood calcium) and 1,25-dihydroxyvitamin D3 participate in the regulation. Pathological conditions appear when the homeostasis of calcium in bone and extracellular fluid is disrupted. Accurate determination of calcium content in blood can provide a scientific basis for the characterization of human physical symptoms. So far, the methods for measuring blood calcium at home and abroad generally include titration, colorimetry, atomic absorption spectrometry, ion selective electrode method, mass spectrometry, fluorescence analysis and so on. The o-cresolphthalein complex colorimetric method is a method with many clinical applications, but the reagents used have poor stability (<8 hours), the interference of magnesium cannot be ruled out, and they are sensitive to carbon dioxide. Atomic absorption spectrometry is the most accurate and sensitive method, but it is difficult to popularize in clinical laboratories because of the high price of the instrument and its maintenance.

Ruthenium terpyridyl ruthenium electrochemiluminescence is an analytical method that has developed relatively rapidly in recent years. It is widely used in the determination of oxalic acid, amino acids and aliphatic amines, immunoassay and nucleic acid analysis. in clinical application. In 1977, Chang, Saji and Bard reported for the first time on page 5399 of volume 99 of J.Am.Chem.Soc. that the electrochemical reaction of ruthenium terpyridine and oxalic acid in an organic solution can produce electrochemiluminescence; in 1981, Rubinstein and Bard in J.Am.Chem.Soc. Volume 103, page 512 reported that the electrochemical reaction of terpyridine ruthenium and oxalic acid in aqueous solution can produce electrochemiluminescence, in 1983 Rubinstein and Bard in Anal.Chem. Volume 55, page 1580 It was reported that ruthenium tertiary pyridine electrochemiluminescence can be used to directly measure oxalic acid in synthetic urine samples. In 1990, Uchikura et al. and Egashira et al. reported on Bunseki Kagaku volume 39, page 323 and Anal.Sci. volume 6, page 903, respectively. Ruthenium tertiary pyridine electrochemiluminescence can be used for direct determination of oxalic acid in urine samples. In 1992, Egashira, Kumasako, Kurauchi and Ohga reported that ruthenium tertiary pyridyl electrochemiluminescence can be used for direct determination on page 713 of Volume 8 of Anal.Sci. Oxalic acid in vegetables. These research results show that terpyridyl ruthenium electrochemiluminescence has good selectivity and sensitivity for the determination of oxalic acid.

The titration method mentioned above to measure blood calcium is to add oxalic acid to the blood to form a precipitate between blood calcium and oxalic acid, wash it several times to remove the remaining oxalic acid and other impurities in the precipitate, and then add sulfuric acid and calcium oxalate to form calcium sulfate precipitate and release it. Oxalic acid, the amount of oxalic acid released by titration with known concentration of potassium permanganate, from which the calcium content was calculated. It can be seen that the operation is complicated and time-consuming, requiring a relatively large number of blood samples.

Contents of the invention

Because ruthenium tertiary pyridine electrochemiluminescence has good selectivity in the determination of oxalic acid, and the concentration of plasma calcium in the human body is generally relatively high, the normal value is 2.25-2.65 mmol/L, which is easy to be precipitated by oxalate, so ruthenium tertiary pyridine electrochemiluminescence has good selectivity. Chemiluminescence can be used for the determination of plasma calcium by directly analyzing the amount of oxalic acid remaining after the formation of calcium oxalate precipitates.

The purpose of the present invention is to provide a method for measuring plasma calcium by ruthenium terpyridyl electrochemiluminescence.

In the present invention, the electrochemiluminescence reagent ruthenium terpyridine and oxalic acid have high luminescence efficiency when the electrochemiluminescence reaction occurs in neutral and weakly acidic solutions, while amino acids, ascorbic acid, etc. in blood can react with ruthenium tertiary pyridine. When the electrochemiluminescence reaction occurs in neutral and weakly acidic solutions, the luminous efficiency is far lower than that of ruthenium terpyridine and oxalic acid; at the same time, the concentration of inherent oxalic acid in blood is very low; Therefore, this method has good selectivity and can meet the needs of measuring plasma calcium to characterize human physiological parameters. The electrochemiluminescence reaction occurs near the electrode surface, and in principle only requires less than 1 microliter of luminescent solution, so it can be measured on very few blood samples. Since the electrochemiluminescence analysis is directly performed on the remaining oxalic acid after the calcium oxalate precipitate is formed, multiple washing and titration processes are omitted, and the analysis speed is significantly improved.

In order to achieve the above object, the technical scheme adopted in the present invention is: first add excess oxalic acid solution (concentration is greater than 5 mmol/liter) in 50～100 microliters of blood plasma, make oxalic acid and blood calcium fully react and generate calcium oxalate, then pass Centrifuge in a centrifuge to make calcium oxalate and macromolecules in the blood sink to the bottom, take the supernatant containing oxalic acid and mix it with ruthenium tertiary pyridine solution with a pH between 5 and 7.5, and then perform electrochemiluminescence analysis, that is, for the generation of oxalic acid The amount of remaining oxalic acid after calcium precipitation is determined by electrochemiluminescence analysis, and the plasma calcium content can be obtained.

In the method for measuring plasma calcium by ruthenium terpyridyl electrochemiluminescence, an acetate buffer can be used when a buffer solution is used.

In the method for measuring blood calcium by ruthenium tertiary electrochemiluminescence, the electrochemiluminescence measurement can be carried out either in a static system or in a flow system.

The method of the invention has the advantages of good selectivity, fast analysis speed, less samples required, relatively cheap analysis instruments and the like. The method of the present invention is quite consistent with the plasma calcium content measured by the most accurate and sensitive atomic absorption spectrometry, and the difference is small. The method of the invention has high reliability and consistency.

Detailed ways

Example 1: Get 100 microliters of plasma and 100 microliters of oxalic acid solution with a concentration of 5 mmol/L and mix evenly so that oxalic acid and blood calcium can fully react, then centrifuge through a centrifuge to make calcium oxalate and macromolecules in blood sink to At the bottom, take the supernatant containing oxalic acid, add ruthenium terpyridyl solution prepared with an acetate buffer solution with a pH value of 5 and mix evenly, then add the mixed solution into the flow injection system for electrochemiluminescence measurement. The calcium content was measured to be 2.478 (±0.053) mmol/L.

Embodiment 2: Carry out comparative test, get 50 microliters of blood plasma and dilute to 100 microliters with the acetate buffer solution of pH value 5.5, mix with 100 microliters concentration and be 5 mmol/liter oxalic acid solution to make oxalic acid and blood calcium fully react, Then centrifuge through a centrifuge to make calcium oxalate and macromolecules in blood sink to the bottom, take the supernatant containing oxalic acid, add ruthenium tertiary pyridine solution prepared with an acetate buffer solution with a pH value of 5.5 and mix evenly, and then mix the mixture Put it into a luminescence cell with a volume of 0.5 ml for static electrochemiluminescence measurement. The calcium content was measured to be 2.496 (±0.054) mmol/L. The calcium content of the same plasma sample measured by atomic absorption spectrometry was 2.531 (±0.029) mmol/L. The plasma calcium content determined by the two methods is quite consistent, and the difference is small.

Embodiment 3: Carry out recovery test, in the blood plasma of 2.48 mmol/liter known calcium amount in 100 microliters, add the calcium of 1.1, 1.5 and 1.9 mmol/liter respectively, make the theoretical value of plasma calcium become 3.58, 3.58, 3.98 and 4.39 mmol/L, and then mixed with the oxalic acid solution with a concentration of 5 mmol/L to make the oxalic acid and calcium react fully, and then centrifuged through the centrifuge to make the calcium oxalate and the macromolecules in the blood sink to the bottom, and take the solution containing To the supernatant of oxalic acid, add ruthenium terpyridyl solution prepared with an acetate buffer solution with a pH value of 7.5 and mix evenly, then put the mixed solution into luminescent cells with a volume of 0.5 ml for static electrochemiluminescence determination. The measured recovery rate is 97.4-102%. Embodiment 4: Carry out repeatability test, get 100 microliters of blood plasma and 100 microliters concentration and be that 5 mmol/liter oxalic acid solution mixes evenly to make oxalic acid and blood calcium fully react, then centrifuge by centrifuge to make calcium oxalate and blood calcium The macromolecules sink to the bottom, take the supernatant containing oxalic acid, add ruthenium terpyridyl solution prepared with an acetate buffer solution with a pH value of 5.5 and mix evenly, then add the mixed solution into the flow injection system for electrochemiluminescence determination. The average value of repeated measurement six times in one day was 2.478 mmol/L, and the coefficient of variation was 2.14%. The average value of repeating the above determination in five days was 2.459 mmol/L, and the coefficient of variation was 3.33%. Facts have proved that the method of the present invention has high reliability and consistency.

Claims (3)

1, a kind of method of measuring plasma calcium by triplex pyridine ruthenium electrochemical luminescence, it is characterized in that, in blood plasma, add concentration greater than 5 mMs/liter excessive oxalic acid solution, make oxalic acid and blood calcium fully react the generation calcium oxalate, undertaken centrifugally making big molsink in calcium oxalate and the blood by hydro-extractor then to the bottom, get the supernatant liquor that contains oxalic acid and mix, carry out electrochemiluminescence analysis then with the terpyridyl ruthenium solution of pH between 5 to 7.5.

2, the method for measuring plasma calcium by triplex pyridine ruthenium electrochemical luminescence as claimed in claim 1 is characterized in that, the buffer solution that uses is acetate buffer solution.

3, tris (bipyridine) ruthenium electrochemiluminescence as claimed in claim 1 is measured the method for blood calcium, it is characterized in that, electrochemiluminescence both can carry out in static systems when measuring, and also can carry out in current system.