A Copper Isotope Reference Material for Biological Matrix Matching and Its Preparation Method

Abstract

The present invention relates to a copper isotope reference material for biological matrix matching and a preparation method thereof, belonging to the technical field of analytical geochemistry. The preparation method of the copper isotope reference material for biological matrix matching is to dissolve NIST SRM 976 in water to obtain a standard solution of copper isotopes, mix agar powder, a polymer dispersant, an ionic dispersant with the standard solution of copper isotopes to obtain a mixed agar solution, heat the mixed agar solution until it is clear and bubble-free and then make it into a semi-solid agar gel, and obtain the product after drying. The preparation method of the copper isotope reference material for biological matrix matching in the present invention is simple and low in cost, which is beneficial to promoting the application of the LA-MC-ICP-MS technology in the analysis of biological samples and is also beneficial to the comparison of data between different laboratories, and this has a great promoting effect on the discipline development of disciplines such as biogeochemistry, environmental science, biology, and medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical geochemistry, and particularly relates to a copper isotope reference material for biological matrix matching and a preparation method thereof. Background Art

[0002] The content, composition and distribution rules of chemical elements and isotopes in animals and plants are one of the important research contents in disciplines such as biogeochemistry, environmental science, biology, and medicine. These elements include essential elements for organisms (N, P, K, Fe, Cu, etc.) and non-essential elements (such as Cr, As, Hg, Tl, Pb, etc.). Essential elements within an appropriate range are necessary conditions for maintaining the normal vital signs of organisms, and exceeding the normal threshold range will cause harm. Non-essential elements do not have a positive promoting effect on the growth, development and metabolism of organisms. Some can be toxic to organisms at trace levels. Since they are difficult to degrade, they can also be amplified by organisms through the food chain, resulting in enrichment in higher-level organisms. Therefore, non-essential elements and essential elements are closely related to environmental safety and agricultural production related to human health. As an essential element for organisms, Cu element is crucial for mitochondrial respiration, enzyme activity, maintenance of protein function and iron metabolism in organisms. The two natural stable isotopes of copper ( 63 Cu and 65 Cu), the isotope abundances in human serum will show measurable changes due to metabolic diseases. Different types of cancers will cause the lighter copper isotope in the patient's blood to be enriched relative to the healthy control group. Therefore, copper isotopes can be used as potential biomarkers for cancer detection and treatment monitoring.

[0003] At present, the most widely used quantitative method for laser ablation (multi-collector) inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) is the external standard calibration method using matrix-matched solid reference materials. This method has low technical difficulty, is relatively easy to implement, and has relatively low costs. However, there is currently no special calibration reference material for the analysis of biological samples by LA-MC-ICP-MS. Each laboratory uses internally synthesized calibration standards when quantitatively analyzing biological samples. Due to the lack of a unified standard for use, it is very difficult to compare data between different experiments. Summary of the Invention

[0004] The first object of the present invention is to provide a copper isotope reference material for biological matrix matching to solve the technical problem of the lack of a unified calibration standard for the detection of copper isotopes in biological samples by LA-MC-ICP-MS.

[0005] The second object of the present invention is to provide a preparation method for a copper isotope reference material for biological matrix matching.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A preparation method of a copper isotope reference material for biological matrix matching. Dissolve NIST SRM 976 in water to obtain a standard solution of copper isotopes. Mix agar powder, a polymer dispersant, an ionic dispersant with the standard solution of copper isotopes to obtain a mixed agar solution. Heat the mixed agar solution until it is clear and bubble-free, and then make it into a semi-solid agar gel. After drying, it is obtained.

[0008] Further, the polymer dispersant is polyvinylpyrrolidone; the ionic dispersant is cetyltrimethylammonium bromide.

[0009] Further, the addition amount of the polymer dispersant is 1-5% of the mass of NIST SRM 976; the addition amount of the ionic dispersant is 3-8% of the mass of NIST SRM 976; the agar powder accounts for 3-5% of the mass of the mixed agar solution.

[0010] Further, the preparation method of the semi-solid agar gel is to first stir at a low speed until the temperature of the mixed agar solution drops to 15-25 °C, then stir at a high speed to remove bubbles, and obtain it after standing for 5-10 minutes.

[0011] Further, the stirring speed of the low-speed stirring is 20-50 rpm, the speed of the high-speed stirring is 300-500 rpm, and the time of the high-speed stirring is 20-30 minutes.

[0012] Further, the pH of the standard solution of copper isotopes is 4-6, and the concentration of NIST SRM 976 in the standard solution of copper isotopes is 80-120 ppm.

[0013] Further, the drying temperature is 30-50 °C, and the drying time is 0.5-1.5 h.

[0014] Further, the heating temperature of the mixed agar solution is 50-70 °C.

[0015] Further, the preparation of the copper isotope reference material for biological matrix matching is carried out in a fume hood.

[0016] A copper isotope reference material for biological matrix matching is prepared by using the above preparation method of the copper isotope reference material for biological matrix matching.

[0017] The beneficial effects of the present invention:

[0018] The preparation method of the copper isotope reference material for biological matrix matching of the present invention is simple and low in cost, which is conducive to promoting the application of LA-MC-ICP-MS technology in the analysis of biological samples and facilitating the comparison of data between different laboratories, and has a great promoting effect on the discipline development of disciplines such as biogeochemistry, environmental science, biology, and medicine.

[0019] The present invention first cools down by low-speed stirring and then removes bubbles by high-speed stirring, which improves the uniformity of copper element distribution and at the same time avoids introducing defoamers into the mixed agar solution, which affects the accuracy of the detection of this copper isotope reference material.

[0020] The present invention makes the copper more evenly dispersed and avoids agglomeration through the steric hindrance effect of the polymer-type dispersant and the electrostatic repulsion of the ionic dispersant. The agar locks the copper element in the network space through a three-dimensional network structure, avoiding the agglomeration and sedimentation of the copper element. The present invention ensures the uniform dispersion and non-sedimentation of the copper element through the compounding of agar, polymer-type polymer, and ionic polymer.

[0021] The long chains in the polymer-type dispersant polyvinylpyrrolidone of the present invention are interspersed in the three-dimensional network formed by agar, making the three-dimensional network structure formed by agar have a stronger gel degree, and the stretching of the long chains occupies a large space, which can achieve a good steric hindrance effect. Description of the Drawings

[0022] Figure 1 Effects on the copper element content and the homogeneity of isotope composition in the copper isotope reference material for biological matrix matching in Examples 1-3 and Comparative Examples 1-3, where (a) is the relative deviation of the copper element content and (b) is the ratio of copper isotopes;

[0023] Figure 2 Effects on the copper element content and the homogeneity of isotope composition in the copper isotope reference material for biological matrix matching in Example 1 and Comparative Examples 4-6, where (a) is the relative deviation of the copper element content and (b) is the ratio of copper isotopes;

[0024] Figure 3 Effects on the copper element content and the homogeneity of isotope composition in the copper isotope reference material for biological matrix matching in Example 1 and Comparative Examples 7-9, where (a) is the relative deviation of the copper element content and (b) is the ratio of copper isotopes;

[0025] Figure 4 For the semi-solid agar gel in Example 1 and Comparative Example 10, where (a) is Example 1 and (b) is Comparative Example 10;

[0026] Figure 5 For the detection results of Application Example 1, where (a) is the content of copper element and (b) is the ratio of copper isotopes;

[0027] Figure 6 For the detection results of Application Example 2, where (a) is the content of copper element and (b) is the ratio of copper isotopes. Specific implementation manners

[0028] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0029] Equipment used in the present invention: An Agilent 7900 inductively coupled plasma mass spectrometer from Agilent Technologies, USA, is used in combination with an NWR 193 nm nanosecond laser from ESI, USA, for elemental content analysis. A Neptune XT multi-collector inductively coupled plasma mass spectrometer from Thermo Fisher Scientific, USA, is used in combination with an NWR 193 nm nanosecond laser from ESI, USA, for copper isotope analysis.

[0030] The purified agar powder used in the present invention is produced by Shanghai Shanpu Chemical Co., Ltd. Ultra-pure water is prepared by a Millipore ultra-pure water system (Suzhou Sains Instruments Co., Ltd.). NIST SRM 976 and NIST SRM 3114 are developed by the National Institute of Standards and Technology, USA.

[0031] In the present invention Figures 1 - 3 The RSD in it represents the relative deviation, and the black bar represents the reference value range (-0.0764 ± 0.0048‰).

[0032] Example 1

[0033] The preparation method of the copper isotope reference material for biological matrix matching in Example 1 includes the following steps:

[0034] S1: Dissolve NIST SRM 976 in water to obtain a standard solution of 9.5 g of copper isotopes. The concentration of NIST SRM 976 in the standard solution of copper isotopes is 100 ppm; the pH of the standard solution of copper isotopes is 5;

[0035] S2: Dissolve polyvinylpyrrolidone and cetyltrimethylammonium bromide in water to obtain a mixed solution. The mass fractions of polyvinylpyrrolidone and cetyltrimethylammonium bromide in the mixed solution are both 0.96 mg / mL. Pipette 50 μL of the mixed solution, and add 0.5 g of purified agar powder and 50 μL of the mixed solution to the standard solution of copper isotopes to obtain a mixed agar solution;

[0036] S3: Heat the mixed agar solution on a 60°C hot plate until the mixed agar solution is clear and free of bubbles;

[0037] S4: Preheat the Teflon plastic target ring placed on a clean glass slide on a hot plate at 60°C. Use a pipette to aspirate 500 μL of the clear and bubble-free mixed agar solution in S3 and inject it into the Teflon plastic target ring; the inner diameter of the Teflon target ring is 15 mm and the height is 3 mm.

[0038] S5: Stir the mixed agar solution at a speed of 20 rpm until the temperature drops to 25°C, then stir at a speed of 350 rpm for 20 min, wait for 5 min, and remove the Teflon plastic target ring to obtain a semi-solid agar gel with a diameter of 15 mm and a height of 3 mm.

[0039] S6: Dry the semi-solid agar gel in an oven at 60°C for 1 h to obtain the product.

[0040] To prevent dust and other impurities in the environment from mixing in, all steps are carried out in a fume hood.

[0041] Example 2

[0042] The preparation method of the copper isotope reference material for biological matrix matching in Example 2 includes the following steps:

[0043] S1: Dissolve NIST SRM 976 in water to obtain a standard solution of copper isotopes with a mass of 9.6 g. The concentration of NIST SRM 976 in the standard solution of copper isotopes is 120 ppm; the pH of the standard solution of copper isotopes is 5.

[0044] S2: Dissolve polyvinylpyrrolidone and cetyltrimethylammonium bromide in water to obtain a mixed solution. The mass fractions of polyvinylpyrrolidone and cetyltrimethylammonium bromide in the mixed solution are 0.92 mg / mL and 0.7 mg / mL respectively. Use a pipette to transfer 50 μL of the mixed solution, and add 0.4 g of purified agar powder and 50 μL of the mixed solution to the standard solution of copper isotopes to obtain a mixed agar solution.

[0045] S3: Heat the mixed agar solution on a hot plate at 50°C until the mixed agar solution is clear and bubble-free.

[0046] S4: Preheat the Teflon plastic target ring placed on a clean glass slide on a hot plate at 50°C. Use a pipette to aspirate 500 μL of the clear and bubble-free mixed agar solution in S3 and inject it into the Teflon plastic target ring; the inner diameter of the Teflon target ring is 15 mm and the height is 3 mm.

[0047] S5: Stir the mixed agar solution at a speed of 30 rpm until the temperature drops to 20°C, then stir at a speed of 300 rpm for 30 min, wait for 10 min, and remove the Teflon plastic target ring to obtain a semi-solid agar gel with a diameter of 15 mm and a height of 3 mm.

[0048] S6: Dry the semi-solid agar gel in an oven at 50 °C for 1 h to obtain the product.

[0049] To prevent dust and other impurities in the environment from mixing in, all steps are carried out in a fume hood.

[0050] Example 3

[0051] The preparation method of the copper isotope reference material for biological matrix matching in Example 3 includes the following steps:

[0052] S1: Dissolve NIST SRM 976 in water to obtain a standard solution of copper isotopes with a mass of 9.7 g. The concentration of NIST SRM 976 in the standard solution of copper isotopes is 80 ppm; the pH of the standard solution of copper isotopes is 6.

[0053] S2: Add polyvinylpyrrolidone and cetyltrimethylammonium bromide to water to obtain a mixed solution. The mass fractions of polyvinylpyrrolidone and cetyltrimethylammonium bromide in the mixed solution are 0.16 mg / mL and 1.24 mg / mL respectively. Pipette 50 μL of the mixed solution, and add 0.3 g of purified agar powder and 50 μL of the mixed solution to the standard solution of copper isotopes to obtain a mixed agar solution.

[0054] S3: Heat the mixed agar solution on a hot plate at 70 °C until the mixed agar solution is clear and free of bubbles.

[0055] S4: Preheat the Teflon plastic target ring placed on a clean glass slide on a hot plate at 70 °C. Use a pipette to suck 500 μL of the clear and bubble-free mixed agar solution in S3 and inject it into the Teflon plastic target ring; the inner diameter of the Teflon target ring is 15 mm and the height is 3 mm.

[0056] S5: Stir the mixed agar solution at a speed of 50 rpm until the temperature drops to 15 °C, then stir at a speed of 500 rpm for 20 min, wait for 10 min, and remove the Teflon plastic target ring to obtain a semi-solid agar gel with a diameter of 15 mm and a height of 3 mm.

[0057] S6: Dry the semi-solid agar gel in an oven at 70 °C for 1 h to obtain the product.

[0058] To prevent dust and other impurities in the environment from mixing in, all steps are carried out in a fume hood.

[0059] Comparative Examples 1 - 3

[0060] The preparation methods of the copper isotope reference materials for biological matrix matching in Comparative Examples 1-3 are substantially the same as those in Example 1. The differences between the preparation methods of the copper isotope reference materials for biological matrix matching in Comparative Examples 1-3 and Example 1 are as follows: in Comparative Example 1, the mass of the standard solution of copper isotope is 9.9 g, and the addition amount of agar powder is 0.1 g; in Comparative Example 2, the mass of the standard solution of copper isotope is 9.8 g, and the addition amount of agar powder is 0.2 g; in Comparative Example 3, the mass of the standard solution of copper isotope is 9.4 g, and the addition amount of agar powder is 0.6 g.

[0061] Comparative Examples 4-6

[0062] The preparation methods of the copper isotope reference materials for biological matrix matching in Comparative Examples 4-6 are substantially the same as those in Example 1. The differences between the preparation methods of the copper isotope reference materials for biological matrix matching in Comparative Examples 4-6 and Example 1 are as follows: in Comparative Example 4, cetyltrimethylammonium bromide is removed and replaced with polyvinylpyrrolidone of the same mass; in Comparative Example 5, polyvinylpyrrolidone is removed and replaced with cetyltrimethylammonium bromide of the same mass; in Comparative Example 6, neither cetyltrimethylammonium bromide nor polyvinylpyrrolidone is added.

[0063] Comparative Examples 7-9

[0064] The preparation methods of the copper isotope reference materials for biological matrix matching in Comparative Examples 7-9 are substantially the same as those in Example 1. The differences between the preparation methods of the copper isotope reference materials for biological matrix matching in Comparative Examples 7-9 and Example 1 are as follows: in Comparative Example 7, the mass fractions of polyvinylpyrrolidone and cetyltrimethylammonium bromide in the mixed solution are 0.01 mg / mL and 0.38 mg / mL respectively; in Comparative Example 8, the mass fractions of polyvinylpyrrolidone and cetyltrimethylammonium bromide in the mixed solution are 0.01 mg / mL and 0.19 mg / mL respectively; in Comparative Example 9, the mass fractions of polyvinylpyrrolidone and cetyltrimethylammonium bromide in the mixed solution are 0.114 mg / mL and 0.19 mg / mL respectively.

[0065] Comparative Example 10

[0066] The preparation method of the copper isotope reference material for biological matrix matching in Comparative Example 10 is substantially the same as that in Example 1. The difference between the preparation method of the copper isotope reference material for biological matrix matching in Comparative Example 10 and Example 1 is that after the mixed agar solution is placed in the Teflon plastic target ring, the Teflon plastic target ring is placed at 25 °C for 10 min, and then the Teflon plastic target ring is removed to obtain a semi-solid agar gel.

[0067] From Figure 1It can be seen that when the agar concentration in the mixed agar solution increases, the uniformity of copper element distribution also increases, and the analysis error of copper isotopes decreases. However, with the further increase of agar concentration, when the agar concentration exceeds 5%, the uniformity of copper element becomes poor, but the analysis error of copper isotopes does not change much. This is mainly because when the agar concentration is too low, the formed gel cannot be well consolidated. The gel strength is poor, and it is difficult to separate from the mold, which is not suitable for LA-MC-ICP-MS analysis. When the agar concentration is too high, the agar solution is too viscous, forming a large number of bubbles, which affects the uniform distribution of copper elements.

[0068] From Figure 2 It can be seen that when either the polymeric dispersant or the ionic dispersant is missing, the distribution effect of copper elements will deteriorate, affecting the uniform distribution of copper elements. Only when the polymeric dispersant and the ionic dispersant coexist in the present invention can the copper elements be evenly distributed without sedimentation. From Figure 3 It can be seen that changing the addition amounts of the polymeric dispersant and the ionic dispersant also has a certain impact on the dispersion of copper elements. When the addition amounts of the polymeric dispersant and the ionic dispersant are too small, the dispersion effect is poor. When the addition amounts of the polymeric dispersant and the ionic dispersant are too large, the excessive dispersant may cause the polymer chains adsorbed on the particle surface to entangle and crosslink with each other, resulting in a poor dispersion effect. Moreover, the dispersant itself also has a certain viscosity. When the amount of the dispersant is too large, the viscosity of the system increases, affecting the dispersion of copper elements. From Figure 4 It can be seen that when the mixed agar solution is directly placed into the Teflon plastic target ring without stirring and defoaming, there are a large number of bubbles in the prepared semi-solid agar gel, which affects the distribution of copper elements.

[0069] The on-machine test process is as follows:

[0070] For the laser ablation system: taking the signal intensity of 238 U + in NIST 612 as more than 300,000 cps, the ablation beam spot size of the laser is 50 microns, the ablation frequency is 5 Hz, and the energy density is 3 joules per square centimeter.

[0071] Actual test: The sample standard cross method (SSB method) is used to verify the accuracy of the data. The copper isotope reference material prepared for biological matrix matching in the present invention is used as the standard sample, and the agar gel standard sample added with NIST SRM 3114 is used as the actual sample to be measured. The experiment is carried out in the order of "standard sample + standard sample + sample + sample + standard sample + standard sample + sample...".

[0072] Data processing: Iolitev4.0 is used for data processing.

[0073] Among them, the calculation formula of copper isotopes is shown in Formula 1. Among them is the copper isotope ratio of the actual sample, is the copper isotope ratio of the standard sample.

[0074] , Formula 1.

[0075] Application Example 1

[0076] Moss growing in a certain abandoned mine pit

[0077] Collect the moss growing around the mine pit, separate and harvest its roots and leaves, repeatedly rinse the soil and gravel adhering to the surface with flowing tap water. After the soil and gravel are rinsed clean, place it in a cool and ventilated place to drain the water. Rinse the roots and leaves of the moss with ultrapure water 5 times again. After draining the water, put it into a paper sample bag. Use an oven at 105 °C to blanch the moss for 30 min, then place it in a fume hood at 80 °C to dry it to a constant weight. After it is completely dried, use an agate mortar to grind and crush the roots and leaves respectively. Pass the ground powder through a 200-mesh sieve for standby.

[0078] Use the method established above to analyze the roots and leaves of the moss, and the test results are as Figure 5 shown. The copper element content in the roots (112 ± 13 ppm) of the moss growing in this abandoned mine pit is significantly higher than that in the leaves (76 ± 8 ppm). The δ 65 Cu NIST976 of the moss roots is 0.08 ± 0.10‰, while the δ 65 Cu NIST976 of the leaves is -0.45 ± 0.10‰. The δ 65 Cu of the roots is similar to the copper isotope composition in the local copper ore, but obvious fractionation of copper isotopes occurs in the leaves, indicating that during the growth process of the moss, the leaves are relatively more enriched in lighter isotopes compared to the roots.

[0079] Application Example 2

[0080] Purple flowering stem near the sewage outlet of a certain factory

[0081] The Brassica parachinensis plants near the sewage outlet of the harvesting factory were collected, and their roots, stems, and leaves were harvested separately. The treatment methods for the roots, stems, and leaves of the Brassica parachinensis plants were similar to those of moss. The soil and gravel adhering to the surface were repeatedly rinsed with flowing tap water. After the soil and gravel were rinsed clean, they were placed in a cool and ventilated place to drain the water. Then, the roots, leaves, and stems of the Brassica parachinensis plants were rinsed 5 times with ultrapure water, and after draining the water, they were put into paper sample bags. The Brassica parachinensis plants were blanched in an oven at 105 °C for 30 min and dried to a constant weight in a fume hood at 80 °C. After complete drying, the roots, leaves, and stems were ground separately using an agate mortar, and the ground powder was passed through a 200-mesh sieve for standby.

[0082] The roots, stems, and leaves of Brassica parachinensis were analyzed using the method established above, and the detection results are as Figure 6 shown. The copper contents in the roots, stems, and leaves of Brassica parachinensis near the sewage outlet of the factory were measured to be 68±8 ppm, 52±6 ppm, and 55±6 ppm, respectively. The δ 65 Cu NIST976 values of the roots, stems, and leaves of Brassica parachinensis were -0.17±0.15‰, -1.13±0.15‰, and -0.77±0.15‰, respectively. The copper isotope composition of the roots of Brassica parachinensis was similar to that of the copper in the soil near the sewage outlet, but significant fractionation occurred in the copper isotope composition of its stems and leaves, with the stems and leaves relatively enriched in lighter copper isotopes. Among them, the fractionation of the copper isotopes in the stems was more obvious, and the copper isotopes in the leaves were more enriched in heavier isotopes than those in the stems.

Claims

1. A preparation method of a copper isotope reference material for biological matrix matching, characterized in that, Dissolve NIST SRM976 in water to obtain a standard solution of copper isotopes. Mix agar powder, a polymer dispersant, an ionic dispersant with the standard solution of copper isotopes to obtain a mixed agar solution. Heat the mixed agar solution until it is clear and free of bubbles, and then make it into a semi-solid agar gel. After drying, it is obtained; the polymer dispersant is polyvinylpyrrolidone; the ionic dispersant is cetyltrimethylammonium bromide; the addition amount of the polymer dispersant is 1-5% of the mass of NIST SRM 976; the addition amount of the ionic dispersant is 3-8% of the mass of NIST SRM 976; the agar powder accounts for 3-5% of the mass of the mixed agar solution; the pH of the standard solution of copper isotopes is 4-6, and the concentration of NIST SRM 976 in the standard solution of copper isotopes is 80-120 ppm.

2. The preparation method of the copper isotope reference material for biological matrix matching according to claim 1, wherein The preparation method of the semi-solid agar gel is to first stir at a low speed until the temperature of the mixed agar solution drops to 15-25 °C, then stir at a high speed to remove bubbles, and obtain it after standing for 5-10 min.

3. The preparation method of the copper isotope reference material for biological matrix matching according to claim 2, characterized in that, The stirring speed of the low-speed stirring is 20-50 rpm, the speed of the high-speed stirring is 300-500 rpm, and the time of the high-speed stirring is 20-30 min.

4. The preparation method of the copper isotope reference material for biological matrix matching according to claim 1, characterized in that, The temperature of the drying is 30-50 °C, and the time of the drying is 0.5-1.5 h.

5. The preparation method of the copper isotope reference material for biological matrix matching according to claim 1, wherein The temperature for heating the mixed agar solution is 50-70 °C.

6. The preparation method of the copper isotope reference material for biological matrix matching according to claim 1, characterized in that, The preparation of the copper isotope reference material for biological matrix matching is carried out in a fume hood.

7. A copper isotope reference material for biological matrix matching, characterized in that, It is prepared by using the preparation method of the copper isotope reference material for biological matrix matching described in claim 1.

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