CN111830116A - Enrichment method, quantitative kit and detection method of silver ions in biological matrix - Google Patents
Enrichment method, quantitative kit and detection method of silver ions in biological matrix Download PDFInfo
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Abstract
The invention discloses a method for enriching silver ions in a biological matrix, a quantitative kit and a detection method, wherein the method for enriching the silver ions comprises the following steps: s1, adding a nitric acid solution I into the biological matrix sample to enable the pH value of the system to be 6.5-8.0; s2, adding a protein solution; s3, adding a nitric acid solution II to enable the pH value of the system to be 1.0-3.0. The silver ion quantitative kit comprises a nitric acid solution I, a nitric acid solution II, a protein solution, a silver standard, a silver control product and an indium internal standard; wherein the concentrations of the nitric acid solution I and the nitric acid solution II are respectively 0.5-2.0%; the protein solution is a fetal bovine serum albumin solution. The silver ion enrichment method provided by the invention realizes the high-efficiency enrichment of silver ions in the biological matrix sample by a protein displacement method, and can effectively improve the accuracy, operation precision and parallelism of the ICP-MS quantitative detection of the silver ions.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a method for enriching silver ions in a biological matrix, a quantitative kit and a detection method.
Background
The medicine with silver ion as the treating component mainly includes dressing, liniment, gel, etc. and is used mainly in surface sterilization, physical neutralization and treating burns. The sterilization mechanism of silver ion is mainly through free Ag+Binding free mercapto group (-SH) of protein in microorganism such as cell, changing surface charge of protein, denaturing protein and coagulating, destroying cell synthetase activity, and killing cell due to loss of division and proliferation ability. The combination of silver ion and protein is reversible, when the thallus loses activity, the silver ion is dissociated from the thallus, and the sterilization activity is repeated, so that the antibacterial effect of the silver ion medicament is durable, and the drug resistance of bacteria is low. Considering that a large dose or long-term use of silver ion drugs causes accumulation of heavy metals and causes acute or chronic poisoning, the use concentration of most of the silver ion drugs approaches to a trace amount (as low as μ g/ml or ng/ml), which undoubtedly poses a challenge to the detection sensitivity and accuracy of the silver ion concentration.
Regarding the determination of silver ion concentration, the Chinese patent 201110204651.9 discloses a detection kit for a solution containing nano-silver and ionic silver, specificallyIt discloses that silver ions and chloride ions in sodium chloride are subjected to chemical reaction to form silver chloride precipitate, and the concentration of the silver ions is indirectly detected by detecting the content of the precipitate, wherein the quantitative concentration is not lower than 0.1 mu g/ml. The method does not need major precision equipment, but the operation precision and accuracy are unknown, and the method cannot be applied to the determination of the concentration of trace silver ions in a complex biological matrix, and some published patent reports are based on TMB (3,3 ', 5, 5' -tetramethyllbenzidine) color development technology to quantitatively analyze the silver ions, but the method has obviously insufficient applicability to the complex matrix. Based on the inorganic metal element characteristics of silver ions, compared with Atomic Absorption Spectrometry (AAS) and Inductively Coupled Plasma atomic emission Spectrometry (ICP-AES), the currently commonly used silver ion detection technology is Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and ICP-MS has the advantages of high sensitivity and wide measurement linear range (10)-6～10-12g/ml) and the like (Zhangjing, Zuoxiao, Madong, ICP-MS analysis research progress of elements in blood and urine [ J].2012,28(6):456-463). However, ICP-MS also reveals some disadvantages in the determination of trace inorganic metal elements over 40 years of practical use: the method is easily subjected to spectral line interference, matrix interference and physical interference, and the accuracy of an analysis result of the method is influenced, so that the sample needs to be pretreated and detection conditions need to be optimized when a complex matrix sample is determined.
The ICP-MS is often applied to content research of iron, manganese, copper, zinc, cadmium, nickel, mercury, arsenic and the like in matrixes such as soil, cosmetics, water, crops, food and the like when detecting inorganic metal elements. Such substrates are characterized by less interference of the substrate and easy separation of the target metal element to be measured. With the development and application of silver ion medicines growing, the ICP-MS for detecting the content of silver ions in a biological matrix is an urgent need, and how to effectively eliminate or reduce the interference of the biological matrix on the detection of the silver ions is a problem to be solved for developing related detection methods and products.
The biological matrix refers to a matrix obtained from a living body, and includes blood (serum, plasma), urine, cerebrospinal fluid and other liquids. Due to the complex composition of the biological matrix, a matrix effect usually exists in the detection process, and the detection accuracy is influenced. The urine belongs to an aqueous solution matrix, the majority of the urine is water, and the urine also contains a small amount of inorganic salt, urea, uric acid and the like, and the obvious difference with biological matrix samples such as whole blood, blood plasma, blood serum and the like is that the urine does not contain or contains extremely low protein and glucose, so that the problem of recovery rate reduction caused by protein-combined silver ions can be avoided. However, in alkaline urine, silver ions with positive charges are very easily adsorbed on a container (such as glass, polypropylene or polystyrene) with negative charges on the surface, and the adsorption is difficult to effectively remove by common ultrasonic treatment and microwave digestion, so that the trouble of low recovery rate is also caused. In addition, since bile excretion is the main mode of eliminating silver, not renal excretion, a very sensitive and efficient pretreatment method for enriching silver ions at extremely low concentration is required if silver is to be detected from urine. Lin YS et al, which detected silver ion concentrations in serum and urine of 16 subjects using ICP-MS, detected 0.67ng/ML silver ions in serum but not in urine, demonstrated that trace amounts of silver ions in urine were extremely difficult to detect (Lin YS, Rothen ML, Milgrom P. Pharmacokinetics of 38% topical silver fluoride in health plus volnents [ J ]. J Am DentAssoc, 2019,150(3): 186-.
Based on the technical advantages and difficulties of ICP-MS detection of silver ions, for element detection in a biological matrix sample, a proper pretreatment method can reduce system blockage, eliminate matrix interference and the like, and the purpose of pretreatment is mainly to enrich the silver ions in the biological matrix and improve the detection accuracy. Therefore, it is necessary to develop a suitable pretreatment method for the analyte bio-matrix sample and a silver ion enrichment method.
At present, the main pretreatment methods for biological aqueous solution matrix (such as urine) include microwave digestion method and direct dilution method. Common digestion reagents for the microwave digestion method comprise nitric acid, hydrogen peroxide, sulfuric acid, hydrochloric acid or perchloric acid, can thoroughly digest samples under a closed high-temperature high-pressure condition, and reduce the loss of volatile elements, but the sample treatment time is long, the steps are complicated, the sample pollution is easy to cause, and the method is not suitable for detection and analysis of a large number of samples; the direct dilution method is simple to operate, and can effectively reduce the pollution between samples or from the outside, but the matrix physicochemical properties between the actual biological matrix sample and the standard solution are greatly different, so that better accuracy is difficult to obtain. Therefore, the pretreatment method and the silver ion enrichment method mainly have the technical problems of poor pretreatment effect of silver ion enrichment, non-specific adsorption and serious residue of silver ions in a matrix and the like, and the ideal effect of complete desorption is difficult to achieve.
The main defects of the pretreatment method are as follows: firstly, the processing method is simple: aiming at the silver ion enrichment pretreatment in the urine sample, a method of directly diluting with nitric acid in one step is mostly adopted, or solid phase extraction is added, but silver ions are easily adsorbed on a tube wall or an extraction column, and the ideal effect of complete desorption is difficult to achieve even if the type and concentration of acid liquor are changed; secondly, the pretreatment effect of silver ion enrichment is poor: aiming at the silver ion enrichment pretreatment in the urine sample, for the purpose of direct dilution by nitric acid, only silver ions on part of tube walls can be desorbed, and still part of the silver ions can not be completely desorbed, the treatment result usually causes obvious residual effect, thereby not only influencing the real measurement value between standard products with different concentrations, but also generating residual interference of a high-concentration sample on a low-concentration sample, and ensuring that the precision and the reproducibility of the actual measurement quantitative result of the silver ions are lower, and the method is difficult to be suitable for the quantitative research of the pharmacokinetics PK/PD of the silver ion medicament. The result of the pretreatment often causes a relatively obvious residual effect, which not only influences the real measured value among different concentration standard substances, but also generates residual interference of a high-concentration sample on a low-concentration sample, so that the precision and the reproducibility of the actual measurement quantitative result of the silver ions are low, and the method is difficult to be applied to the quantitative research of the pharmacokinetics PK/PD of the silver ion medicament. Therefore, it is of great importance to develop an improved method for enriching silver ions in biological matrix and a kit thereof.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a method for enriching silver ions in a biological matrix, namely a protein replacement method, which can improve the enrichment efficiency of the silver ions in the biological matrix through the protein replacement method, is used for depolymerization and desorption of the silver ions in a biological matrix sample, and solves the problems of nonspecific adsorption and serious residue of the silver ions in the matrix.
The invention also provides a quantitative kit for silver ions in the biological matrix.
The invention also provides a quantitative detection method for silver ions in the biological matrix.
A method for enriching silver ions in a biological matrix according to an embodiment of the first aspect of the present invention comprises the steps of:
s1, adding a nitric acid solution I into the biological matrix sample to enable the pH value of the system to be 6.5-8.0, and obtaining a mixed solution I;
s2, adding a protein solution to obtain a mixed solution II;
s3, adding a nitric acid solution II to enable the pH value of the system to be 1.0-3.0.
The silver ion enrichment method provided by the embodiment of the invention has at least the following beneficial effects: the invention enriches the silver ions in the urine by a protein displacement method, utilizes the polymerization capability of protein-silver ion chelate to be stronger than the nonspecific adsorption capability of the silver ions and the tube wall, and simultaneously, the chelate can realize polymerization and depolymerization by changing pH so as to realize the high-efficiency enrichment of the silver ions in the biological matrix. According to some embodiments of the invention, the biological matrix is an alkaline biological matrix, including but not limited to urine; preferably urine. The solution according to the invention is suitable for most biological matrices, in particular for basic biological matrices with low or no protein content. In the invention, the pH value of the urine is 8.0-8.5.
According to some embodiments of the invention, the mass concentration of the nitric acid solution I and the nitric acid solution II is 0.5 to 2.0 percent; the concentration of the nitric acid solution II is higher than that of the nitric acid solution I.
Preferably, the mass concentration of the nitric acid solution I is 0.5-1.0%; more preferably, the mass concentration of the nitric acid solution I is 0.7%.
Preferably, the mass concentration of the nitric acid solution II is 0.7-1.5%; more preferably, the mass concentration of the nitric acid solution II is 1.0%.
According to some embodiments of the invention, the volume of the bio-matrix sample is greater than the volume of the nitric acid solution I, and the volume ratio of the bio-matrix sample to the nitric acid solution I is (1.5-2.5): 1.
preferably, the volume ratio of the biological matrix sample to the nitric acid solution I is 2: 1.
according to some embodiments of the invention, the protein in the protein solution is a common protein including at least one of Human Serum Albumin (HSA), Bovine Serum Albumin (BSA), Ovalbumin (OVA) and fetal Bovine Serum Albumin, and more preferably, the protein solution is a fetal Bovine Serum Albumin solution.
According to some embodiments of the invention, the volume of the mixed solution I is greater than the volume of the protein solution, and the volume ratio of the mixed solution I to the protein solution is (2-4): 1.
preferably, the volume ratio of the mixed solution I to the protein solution is 3: 1.
according to some embodiments of the invention, the volume of the mixed solution II is less than the volume of the nitric acid solution II, and the volume ratio of the mixed solution II to the nitric acid solution II is 1: (3-5).
Preferably, the volume ratio of the mixed solution II to the nitric acid solution II is 1: 4.
further, the silver ion enrichment method further comprises the following steps: and (4) transferring the solution obtained in the step S3 to a low adsorption container by adopting a separation technology, and carrying out sample injection detection.
Preferably, the separation technique is shaking; the shaking time was 3 min.
According to some embodiments of the invention, the method specifically comprises the following steps:
1) firstly, mixing a urine sample with a small amount of dilute nitric acid in a certain ratio (the initial volume of the urine sample is not exceeded, and the optimal volume ratio is urine: the diluted nitric acid is 2:1, the optimal concentration of the diluted nitric acid is 0.7 percent), the pH value of urine is changed to be alkalescent or neutral (the pH value is 6.5-8.0), and most of silver ions on the tube wall of the desorption container are desorbed;
2) then adding a certain amount of protein solution (the optimal volume ratio is that urine dilute nitric acid is mixed, namely protein is 3:1, and the optimal protein is fetal calf serum), utilizing the advantage that the adsorption acting force of metal substances such as protein and silver ions under the weak alkaline or neutral environment is far greater than that of the adsorption acting force of the container tube wall and the silver ions, and one protein molecule can be combined with n metal substances such as silver ions to replace and combine the silver ions on the container tube wall and partially dissociated in the solution to form a protein-silver ion chelate;
3) then adding diluted nitric acid with proper multiplying power (the optimal volume ratio is protein-silver ion chelate: 1:4 of dilute nitric acid, wherein the optimal concentration of the dilute nitric acid is 1.0%), changing the pH of urine into acidity, depolymerizing a protein-silver ion chelate to form easily soluble nitrate similar to silver nitrate, and blocking reabsorption of silver ions and the tube wall by the dissociated protein due to steric hindrance to obtain all free silver ions;
4) and finally, transferring the free silver ions existing in the multiplying power dilute nitric acid system into a low adsorption container by adopting a proper separation technology (the optimal separation mode is oscillation, and the optimal time is 3min), and carrying out sample injection detection.
According to the silver ion quantitative kit of the embodiment of the second aspect of the invention, the silver ion quantitative kit comprises a nitric acid solution I, a nitric acid solution II, a protein solution, a silver standard, a silver quality control product and an indium internal standard product; wherein the concentrations of the nitric acid solution I and the nitric acid solution II are respectively 0.5-2.0%; the concentration of the nitric acid solution II is higher than that of the nitric acid solution I; the protein solution is a fetal bovine serum albumin solution.
The silver ion quantification kit provided by the embodiment of the invention has at least the following beneficial effects: the kit optimizes the stable quantitative method parameter result, obtains the reasonable linear range, standard substance and quality control substance setting required by the quantitative kit, verifies the quantitative sensitivity, accuracy and precision, stability, interference test and other performances, is preliminarily applied to quantitative test and evaluation in a small amount of actual biological matrix samples, meets the quality standard, has the preliminary prototype of the quantitative kit product, and has great market prospect and value.
According to some embodiments of the invention, the silver ion quantification kit is detected by ICP-MS, and the silver standard comprises: 1) concentration of silver standard curve working solution: 300. 600, 1200, 2400, 4800, 7200, 10000 ng/mL-1The solvent is an indium-containing internal standard 1000 ng/mL-10.7% nitric acid; 2) silver standard urine sample concentration: 15. 30, 60, 120, 240, 360, 500 ng/mL-1The solvent is clinically collected urine of healthy subjects;
the silver quality control product comprises: 1) silver quality control working solution: 300. 700, 3000, 5000, 7600 ng.mL-1The solvent is an indium-containing internal standard 1000 ng/mL-10.7% nitric acid; 2) silver quality control urine sample: 15. 35, 150, 250, 380 ng/mL-1The solvent is urine of a healthy subject collected clinically.
Further, the mass concentration of the nitric acid solution I is 0.7%; the mass concentration of the nitric acid solution II is 1.0%.
Further, the stock solution concentration of the silver standard substance is 1000 mug. mL-1；
Further, the working solution concentration of the indium internal standard is 1000 ng-mL-1The solvent was 0.7% nitric acid.
According to some embodiments of the present invention, the method for using the silver ion quantification kit comprises preparation of detection reagents and samples, standard curve drawing, sample detection and the like.
Further, the preparation method of the ICP-MS detection reagent and the sample comprises the following steps:
silver standard curve working solution: 300 ng/mL-1(STD1)、600ng·mL-1(STD2)、1200ng·mL-1(STD3)、2400ng·mL-1(STD4)、4800ng·mL-1(STD5)、7200ng·mL-1(STD6)、10000ng·mL-1(STD7) solvent 0.7% nitric acid (containing internal standard);
silver standard urine sample: 0 ng/mL-1(S0)、15ng·mL-1(S1)、30ng·mL-1(S2)、60ng·mL-1(S3)、120ng·mL-1(S4)、240ng·mL-1(S5)、360ng·mL-1(S6)、500ng·mL-1(S7), the solvent is clinically collected urine of a healthy subject;
silver quality control working solution: 300 ng/mL-1(SLLOQ)、700ng·mL-1(SLQC)、3000ng·mL-1(SLMQC)、5000ng·mL-1(SHMQC)、7600ng·mL-1(SHQC) in 0.7% nitric acid (containing internal standard);
silver quality control urine sample: 15 ng/mL-1(LLOQ)、35ng·mL-1(LQC)、150ng·mL-1(LMQC)、250ng·mL-1(HMQC)、380ng·mL-1(HQC), the solvent is clinically collected urine from healthy subjects;
indium internal standard working solution: 1000 ng/mL-1The solvent was 0.7% nitric acid.
Further, the ICP-MS detection parameters used were: ICP-MS Mass spectrometer (model 7900, Agilent), with ICP as the ion source, 2 ℃ for the temperature of the atomization Chamber and 15 L.min for the flow rate of the support-1The sampling depth is 8mm, the number of single peak collecting points is 3, the lifting time is 20s, the stabilization time is 10s, the atom collecting mass silver of the object to be detected is 107, the atom collecting mass indium of the internal standard object is 115, the integration time is 0.1s, the outer wall needle washing mode is to wash 30s by using 5% nitric acid solution, the inner wall needle washing mode is to wash 10s by using 0.5% nitric acid solution firstly, and then wash 50s by using ultrapure water.
The silver ion quantitative detection method comprises the steps of pretreating a protein-containing biological matrix sample to obtain a sample to be detected and quantitatively detecting the sample to be detected by detection equipment, wherein the pretreatment comprises the following steps:
s01, adding a nitric acid solution I into the sample to enable the pH value of the system to be 6.5-8.0;
s02, adding a protein solution;
s03, adding a nitric acid solution II to enable the pH value of the system to be 1.0-3.0;
and S04, separating and transferring to obtain a sample to be detected.
The quantitative detection method for silver ions has at least the following beneficial effects: the optimized biological matrix sample pretreated by the protein replacement method is used for quantitative detection of silver ions, so that the recovery rate of the silver ions is kept at 100 +/-10%, and the accuracy and precision reach the standard. Solves the practical technical problems that the accuracy cannot be ensured by nonspecific adsorption of silver ions and tube walls and direct desorption of nitric acid in a biological matrix sample.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram of the basic principle of "protein replacement method" for enriching silver ions in urine according to an embodiment of the present invention;
FIG. 2 is a quantitative standard curve diagram of ICP-MS for silver ions in urine samples in example 3 of the present invention.
Detailed Description
In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.
The most key innovation of the invention is as follows: provides an efficient silver ion enrichment method, mainly aiming at alkaline, especially aqueous solution type biological matrixes (such as urine), and improving the overall recovery rate and the quantitative effect of silver ions in the biological matrixes by a protein replacement method.
In the invention, because the pH of the urine is alkaline, silver ions in the urine are very easy to nonspecifically adsorb on the tube wall of the container in an alkaline environment, and metal substances such as silver ions and the like can be partially desorbed by changing the pH value through an acidic substance, but the free silver ions cannot be completely obtained by directly adding a large amount of acidic substances, so that the recovery rate of the silver ions in direct detection or common acid treatment is low. Therefore, it is a problem to be solved to ensure that the silver ions are desorbed into free form as much as possible and are not nonspecifically adsorbed again with the tube wall after separation. Based on the problems, the invention provides a protein displacement method for enriching silver ions in a urine sample.
The silver ion enrichment method adopted by the invention is a protein displacement method, and mainly utilizes the fact that the polymerization capacity of a protein-silver ion chelate is stronger than the nonspecific adsorption capacity of silver ions and a pipe wall, and the chelate can realize polymerization and depolymerization through the change of pH, so that the silver ions in a biological matrix can be efficiently enriched. The basic principle of the protein displacement method for enriching silver ions in urine is shown in figure 1, and the specific steps and action principle are as follows:
1) firstly, mixing a urine sample with a small amount of dilute nitric acid in a certain ratio (the initial volume of the urine sample is not exceeded, and the optimal volume ratio is urine: the diluted nitric acid is 2:1, the optimal concentration of the diluted nitric acid is 0.7 percent), the pH value of the urine is changed to be alkalescent or neutral (the pH value has a good effect within the range of 6.5-8.0, the actual pH value of the embodiment comprises 6.6, 6.8, 7.3 and 7.7), and most of silver ions on the tube wall of the desorption container are desorbed;
2) then adding a certain amount of protein solution (the optimal volume ratio is that urine dilute nitric acid is mixed, namely protein is 3:1, and the optimal protein is fetal calf serum), utilizing the adsorption acting force of metal substances such as protein and silver ions under the weak alkaline or neutral environment, which is far greater than the advantage of the adsorption acting force of the container tube wall and the silver ions, wherein one protein molecule can combine n metal substances such as silver ions, and replace and combine part of silver ions which are dissociated on the container tube wall in the solution to form a protein-silver ion chelate;
3) then adding diluted nitric acid with proper multiplying power (the optimal volume ratio is protein-silver ion chelate: 1:4 of dilute nitric acid, wherein the optimal concentration of the dilute nitric acid is 1.0%), changing the pH of urine to be 1.5 (the pH has a good effect within the range of 1.0-3.0, and when the pH is about 1.5, the depolymerization effect of protein-silver ions is optimal), depolymerizing a protein-silver ion chelate to form easily-soluble nitrate similar to silver nitrate, and in addition, the dissociated protein blocks the reabsorption of silver ions and the tube wall due to steric hindrance to obtain all free silver ions;
4) and finally, transferring the free silver ions existing in the multiplying power dilute nitric acid system into a low adsorption container by adopting a proper separation technology (the optimal separation mode is oscillation, and the optimal time is 3min), and carrying out sample injection detection.
The invention provides a protein replacement method by utilizing the principle that the binding force of silver ions with protein is stronger than the binding force with the wall of a container when the pH is alkaline, alkalescent or neutral, and metal chelates such as protein-silver ions are bound under alkaline condition and completely dissociated under acidic condition; the purpose of replacing silver ions is achieved by adding protein to polymerize the silver ions, the formed protein-silver ion chelate is dissociated after the pH value is changed, free silver ions are replaced, and the polymerization and depolymerization of the silver ions are realized; meanwhile, the chelate can realize polymerization and depolymerization through the change of pH, and the efficient enrichment of silver ions in the biological matrix is realized by adjusting the pH, the protein proportion in protein replacement and the pH change after protein replacement through optimizing the adding proportion of nitric acid before protein replacement; the method can also improve the accuracy, operation precision and parallelism of the ICP-MS quantitative detection of the silver ions as a sample pretreatment method.
EXAMPLE 1 method for enriching silver ions in urine sample
The pretreatment method (protein precipitation method) of the urine sample comprises the following specific steps: taking 200 μ L of blank urine, adding 100 μ L of 0.7% nitric acid, adding 100 μ L fetal calf serum, mixing well, adding 1600 μ L of 1% nitric acid, shaking for 3min, sucking 100 μ L into high density polypropylene EP tube, and centrifuging for 5min at 18000 g. The invention enriches the silver ions in the urine by a protein displacement method, utilizes the polymerization capability of protein-silver ion chelate to be stronger than the nonspecific adsorption capability of the silver ions and the tube wall, and simultaneously, the chelate can realize polymerization and depolymerization by changing the pH value. The high-efficiency enrichment of silver ions in the biological matrix sample is realized by adjusting the pH, the protein proportion in protein replacement and the pH change after protein replacement by optimizing the adding proportion of nitric acid before protein replacement.
The solution containing silver ions is treated by a protein replacement method and transferred to a low-adsorption container (the optimal low-adsorption container is made of high-density polypropylene), so that the situation that the final recovery rate is influenced because the enriched free silver ions are adsorbed on the tube wall of the container can be effectively avoided.
The optimized urine sample pretreated by the protein replacement method is used for carrying out ICP-MS quantitative detection on silver ions, so that the recovery rate of the silver ions can be kept at 100 +/-10%, and the accuracy and precision reach the standard. The method solves the practical technical problems that the accuracy cannot be ensured by nonspecific adsorption of silver ions and tube walls and direct desorption of nitric acid in the urine sample.
Example 2ICP-MS quantification kit and method of use thereof
1. The kit comprises the following components: the kit comprises a silver standard substance, a silver quality control substance, an indium internal standard substance, a high-density polypropylene EP tube, a nitric acid solution I (0.7%), a nitric acid solution II (1%) and fetal calf serum.
The use of the kit and the preparation concentration of the working solution are as follows:
silver standard stock solution (CSS): 1000. mu.g/mL-1，
Silver standard curve working solution: 300 ng/mL-1(STD1)、600ng·mL-1(STD2)、1200ng·mL-1(STD3)、2400ng·mL-1(STD4)、4800ng·mL-1(STD5)、7200ng·mL-1(STD6)、10000ng·mL-1(STD7) solvent 0.7% nitric acid (containing internal standard).
Silver standard urine sample: 0 ng/mL-1(S0)、15ng·mL-1(S1)、30ng·mL-1(S2)、60ng·mL-1(S3)、120ng·mL-1(S4)、240ng·mL-1(S5)、360ng·mL-1(S6)、500ng·mL-1(S7), the solvent is clinically collected urine from healthy subjects.
Silver quality control working solution: 300 ng/mL-1(SLLOQ)、700ng·mL-1(SLQC)、3000ng·mL-1(SLMQC)、5000ng·mL-1(SHMQC)、7600ng·mL-1(SHQC), solvent 0.7% nitric acid (containing internal standard).
Silver quality control urine sample: 15 ng/mL-1(LLOQ)、35ng·mL-1(LQC)、150ng·mL-1(LMQC)、250ng·mL-1(HMQC)、380ng·mL-1(HQC), solvent is clinically collected healthy test urine.
Indium internal standard working solution: 1000 ng/mL-1The solvent was 0.7% nitric acid.
The ICP-MS detection parameters used by the kit of the embodiment are as follows: ICP-MS Mass spectrometer (model 7900, Agilent), with ICP as the ion source, 2 ℃ for the temperature of the atomization Chamber and 15 L.min for the flow rate of the support-1The sampling depth is 8mm, the number of single peak collecting points is 3, the lifting time is 20s, the stabilization time is 10s, the atom collecting mass silver of the object to be detected is 107, the atom collecting mass indium of the internal standard object is 115, the integration time is 0.1s, the outer wall needle washing mode is to wash 30s by using 5% nitric acid solution, the inner wall needle washing mode is to wash 10s by using 0.5% nitric acid solution firstly, and then wash 50s by using ultrapure water.
The kit comprises a quantitative result judgment standard: coefficient of correlation r2Not less than 0.9801 or r not less than 0.9900; the calculated-back concentration of the corrected standard sample is within the range of 85-115% of the marked value, and the lower limit of the quantification is within the range of 80-120% of the marked value; at least 75% of the calibration standards and a minimum of 6 concentration levels should meet the above criteria.
2. Urine pretreatment method specified in kit (same as the silver ion enrichment method in example 1)
Taking 200 μ L of blank urine, adding 100 μ L of 0.7% nitric acid (containing 400 ng. mL)-1Internal standard substance indium), then 100 mul fetal calf serum is added, after uniform mixing, 1600 mul 1% nitric acid is added, oscillation is carried out for 3min, 100 mul is absorbed into a high-density polypropylene EP tube, and 18000g is centrifuged for 5 min.
3. Preparation of silver standard curve solution in kit
Taking silver standard solution (national nonferrous metals electronic material analysis and test center, calibration concentration is 1000 mug. mL)-1) Appropriate amount to polypropylene EP tube, and storing at room temperature as stock solution CSS. Then 0.7% nitric acid is used as a solvent, and 7 silver standard curve working solutions with the concentrations of 300 ng/mL are obtained through serial dilution-1(STD1)、600ng·mL-1(STD2)、1200ng·mL-1(STD3)、2400ng·mL-1(STD4)、4800ng·mL-1(STD5)、7200ng·mL-1(STD6)、10000ng·mL-1(STD7)。
In clinicCollecting urine of healthy subject as solvent, further diluting silver standard curve working solution at a ratio of 1:20 to obtain 7 silver standard urine samples with concentration of 15 ng/mL-1(S1)、30ng·mL-1(S2)、60ng·mL-1(S3)、120ng·mL-1(S4)、240ng·mL-1(S5)、360ng·mL-1(S6)、500ng·mL-1(S7), and further, 0 ng. mL is added-1(S0, no silver standard curve working solution was added to normal human urine) and these serially diluted bio-matrix-containing samples were used as x-axis standard concentrations for the quantitative standard curves.
The preparation of the silver standard curve solution is carried out through the stage dilution of stock solution → standard curve working solution → standard urine sample, the solvent is respectively changed into 6.3 percent nitric acid → 0.7 percent dilute nitric acid → urine, and the 0.7 percent dilute nitric acid can not only maintain the solution system to be faintly acid, prevent the pipe wall of a silver ion adsorption container, but also can properly depolymerize silver ions and protein polymers. The treatment avoids silver ion nonspecific adsorption caused by drastic change of pH value due to direct transition from nitric acid with higher concentration to biological matrix, ensures the recovery rate and accuracy of the standard substance, can reduce the span of dilution each time (no more than 100 times, and dilution within 20 times is optimal), and effectively improves the repeatability and accuracy of personnel operation.
4. Preparation of silver control solution in kit
Taking silver standard solution (national nonferrous metals electronic material analysis and test center, calibration concentration is 1000 mug. mL)-1) The appropriate amount is added into a polypropylene EP pipe, and the pipe is preserved at room temperature to be used as stock solution QSS. Then using 0.7% nitric acid as solvent, and making serial dilution to obtain 5 silver quality control working solutions whose concentrations are respectively 300 ng.mL-1(SLLOQ)、700ng·mL-1(SLQC)、3000ng·mL-1(SLMQC)、5000ng·mL-1(SHMQC)、7600ng·mL-1(SHQC)。
Clinically collected urine of healthy subjects is taken as a solvent, and the silver quality control working solution is further diluted in series according to the ratio of 1:20 to obtain 5 silver quality control urine samples with the concentration of 15ng & mL respectively-1(LLOQ)、35ng·mL-1(LQC)、150ng·mL-1(LMQC)、250ng·mL-1(HMQC)、380ng·mL-1(HQC), these serially diluted bio-matrix containing samples, as quantitative quality control standards.
The preparation of the silver quality control solution is carried out through the stage dilution of stock solution → quality control working solution → quality control urine sample, the solvent is respectively changed into 6.3 percent nitric acid → 0.7 percent dilute nitric acid → urine, and the 0.7 percent dilute nitric acid can not only maintain the solution system to be faintly acid, prevent the silver ion from adsorbing the tube wall of the container, but also can properly depolymerize the silver ion and the protein polymer. The treatment avoids silver ion nonspecific adsorption caused by drastic change of pH value due to direct transition from nitric acid with higher concentration to biological matrix, ensures the recovery rate and accuracy of the standard substance, can reduce the span of dilution each time (no more than 100 times, and dilution within 20 times is optimal), and effectively improves the repeatability and accuracy of personnel operation.
5. Preparation of internal standard working solution in kit
Taking indium standard solution (national nonferrous metals electronic material analysis and test center, calibration concentration is 1000 mug. mL)-1) Appropriate amount to polypropylene EP tube, and storing at room temperature as stock solution ISS. Then diluting with 0.7% nitric acid solution to obtain indium internal standard transition liquid (10000ng & mL)-1). Further diluting with 0.7% nitric acid solution to obtain indium internal standard working solution (1000 ng. mL)-1)。
6. Result calculation and statistical mode of kit
And (3) feeding the sample into a mass spectrometry system for analysis, recording the response of the substance to be detected and the internal standard, and calculating the response ratio of the drug to be detected and the internal standard. Taking the response ratio (Y) of silver and the internal standard as the ordinate, taking the concentration (X) of the series of standard urine samples as the abscissa, and using the weight factor (1/X)2) And performing linear regression by a least square method to obtain a linear regression equation and a correlation coefficient. Substituting the response ratio of the substance to be tested and the internal standard in the series of standard urine samples into a linear regression equation for calculation, and comparing the concentration value obtained by testing the series of standard curve concentration samples with the marked concentration value.
The kit of the invention at least has the following beneficial effects: the invention realizes the aim of developing a quantitative kit on the basis of method parameter optimization and verification results. The experimenter optimizes the stable quantitative method parameter result through a large amount of experimental groping, obtains the reasonable linear range, the standard product and the quality control product setting required by the quantitative kit, verifies the quantitative sensitivity, the accuracy and the precision, the stability, the interference test and other performances, and is preliminarily applied to the quantitative test and the evaluation in a small amount of actual biological matrix samples, and the works meet the quality standard and have the preliminary prototype of the quantitative kit product.
At present, the existing silver ion quantitative kit in the market mainly detects water quality and soil matrix, and lacks products for biological matrix related quantitative detection, and the silver ion quantitative kit of the non-biological matrix cannot be used for detecting biological matrix samples, so that the development of the silver ion quantitative kit suitable for different biological matrices has great market prospect and value.
Example 3 kit-related index validation test
1. Drawing of standard curve
A standard curve urine series of concentration samples and quality control samples were prepared as in example 2. And (3) feeding the sample into a mass spectrometry system for analysis, recording the response of the substance to be detected and the internal standard, and calculating the response ratio of the substance to be detected and the internal standard. Taking the response ratio (Y) of silver and the internal standard as the ordinate, taking the concentration (X) of the series of standard urine samples as the abscissa, and using the weight factor (1/X)2) And performing linear regression by a least square method to obtain a linear regression equation and a correlation coefficient. Substituting the response ratio of the substance to be tested and the internal standard in the series of standard urine samples into a linear regression equation for calculation, and comparing the concentration value obtained by testing the series of standard curve concentration samples with the marked concentration value.
The quantitative standard curve of silver ions in urine is shown in figure 2, and the result shows that the kit is 15-500 ng.mL-1The linear equation in the range of y is 0.0059X +3.9533E-007, r is 1.0000, the linear acceptance criterion is met, and the linear quantification effect is good.
2. Verification of accuracy and precision
The preparation concentrations are LLOQ, LQC, LMQC, HMQC and HQC(15、35、150、250、380ng·mL-1) The quality control standard urine samples of (1) were processed according to the sample pretreatment method in example 1 and the application method of the reagent kit in example 2, and then introduced into a mass spectrometry system for analysis. And recording the ratio of the response of the object to be measured and the internal standard, substituting the ratio into the corresponding linear regression equation, calculating the concentration of each sample, comparing the concentration with the sample mark value, and calculating the internal precision and accuracy according to the concentration.
The above steps are carried out for at least 2 days, and 3 batches of the extract have LLOQ, LQC, LMQC, HMQC and HQC (15, 35, 150, 250, 380 ng. mL)-1) And substituting the standard urine sample into a corresponding linear regression equation to calculate the concentration measurement value of each sample, and calculating the batch accuracy and precision of 3 batches of samples at each concentration level according to the concentration measurement value.
Acceptance criteria: the accuracy of the mean value of the measured concentration of the lower quantitative limit should be within 80% -120% of the indicated value, and the accuracy deviation of at least 2/3 samples is within +/-20% of the indicated value; the accuracy of the mean values of the concentrations measured for other quality control concentration standard urine samples should be within 85% -115% of the indicated values, and the accuracy deviation of each concentration of at least 2/3 samples is within ± 15% of the indicated values; the quantitative lower limit precision in batch and between batches is not more than 20%, and the precision in batch and between batches with other quality control concentrations is not more than 15%.
The accuracy and precision verification experiment results of the invention are shown in table 1:
TABLE 1 kit accuracy and precision verification results
The results in table 1 show that the accuracy deviation of the kit in 5 silver quality control products with different concentrations after repeated determination for 6 times does not exceed-2.61% -5.23% of the indicated value, the accuracy deviation accords with the acceptance standard within +/-15%, and the accuracy of the method and the kit is qualified. The silver quality control product under each concentration has the precision CV percent of 0.76-1.93 percent in batch and between batches according to the 6 times of measurement results, the precision CV percent meets the acceptance standard of less than or equal to 15 percent, and the precision of the method and the kit is qualified.
The invention creates a protein replacement method by utilizing the principle that the binding force of silver ions with protein is stronger than that with the wall of a container when the pH is alkaline, alkalescent or neutral, and metal chelates such as protein-silver ions are bound under alkaline and are completely dissociated under acidic conditions. The purpose of silver ion replacement is achieved by adding protein to polymerize silver ions, the formed protein-silver ion chelate is dissociated after pH changes, free silver ions are replaced, polymerization and depolymerization of the silver ions are achieved, low-adsorption materials (preferably high-density polypropylene EP tubes) are used in an auxiliary mode, and the results of examples prove that the silver ion ICP-MS quantitative result of the pretreatment method is high in accuracy, high in operation precision and good in parallelism.
3. Verification of room temperature processing stability
Preparation is low (15 ng. mL)-1LQC), high concentration (380 ng. mL)-1HQC) quality control standard urine samples are respectively processed according to the method of 5.4-5.6, frozen at the temperature of-20 ℃ for a period of time, taken out and unfrozen at room temperature, placed at the room temperature for about 24 +/-5 hours, and injected into a mass spectrum system for analysis. And recording the response ratio of the object to be detected and the internal standard, substituting the response ratio into the corresponding linear regression equation, calculating the concentration of each sample, comparing the concentration with the sample mark value, and inspecting the processing stability of the urine sample at room temperature according to the concentration.
Acceptance criteria: the deviation of the accuracy of the mean value of the measured values of the standard urine sample from the indicated value at each concentration level should be within + -15%, and the accuracy of the sample at each concentration of at least 2/3 should be within + -15% of the indicated value, and the coefficient of variation of the measured values of the sample at the same concentration should not exceed 15%.
The results of the room temperature stability verification experiment of the present invention are shown in table 2:
table 2 sample pretreatment room temperature stability verification results
The results in table 2 show that the silver quality control products with different concentrations of low silver and high silver of the kit are processed for about 24 plus or minus 5 hours (actual processing is 19 hours) at room temperature, the accuracy deviation of 6 repeated determinations does not exceed-0.71-6.76 percent of the indication value, and the accuracy deviation accords with the acceptance standard within plus or minus 15 percent; the silver quality control product under each concentration has the precision CV percent of 0.71-1.34 percent of the result of 6 times of measurement, which meets the acceptance standard of less than or equal to 15 percent, and the stability of the sample pretreatment at room temperature is qualified.
The investigation time of the room temperature stability reaches 19h, which indicates that the silver ions in the urine can be stably kept at room temperature by the protein replacement method and the low-adsorption container (preferably a high-density polypropylene EP tube), and the accuracy of quantitative measurement and the precision of repeated measurement are not influenced. The silver ion enrichment method and the ICP-MS quantitative kit solve the problems of residual interference and nonspecific adsorption commonly existing in the existing silver ion quantitative technology or products, and have obvious technical innovation advantages and practical application value.
According to the direct dilution method reported by literatures and the like and the addition of different surfactants, the experiment personnel verify that the replacement of the surfactant can seriously interfere the detection system of the mass spectrum to cause the background interference of silver ion detection, and the direct dilution method can still generate obvious effects of tube wall adsorption and silver ion residue.
Compared with a direct dilution method, the method for pre-treating the biological matrix and enriching the silver ions can obviously reduce the tube wall adsorption and the residual effect of the silver ions and improve the accuracy and precision of ICP-MS quantitative detection of the silver ions.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for enriching silver ions in a biological matrix is characterized by comprising the following steps:
s1, adding a nitric acid solution I into the biological matrix sample to obtain a mixed solution I, wherein the pH value of the system is 6.5-8.0;
s2, adding a protein solution to obtain a mixed solution II;
s3, adding a nitric acid solution II to enable the pH value of the system to be 1.0-3.0.
2. The silver ion enrichment method of claim 1, wherein the biomatrix is an alkaline biomatrix; preferably urine.
3. The silver ion enrichment method of claim 1, wherein the mass concentrations of the nitric acid solution I and the nitric acid solution II are 0.5-2.0% respectively; the concentration of the nitric acid solution II is higher than that of the nitric acid solution I.
4. The silver ion enrichment method according to claim 1, wherein the volume of the bio-matrix sample is larger than the volume of the nitric acid solution I, and the volume ratio of the bio-matrix sample to the nitric acid solution I is (1.5-2.5): 1.
5. the silver ion enrichment method of claim 1, wherein the protein in the protein solution comprises at least one of human serum albumin, bovine serum albumin, ovalbumin, and fetal bovine serum albumin; preferably fetal bovine serum albumin.
6. The silver ion enrichment method of claim 1, wherein the volume of the mixed solution I is larger than that of the protein solution, and the volume ratio of the mixed solution I to the protein solution is (2-4): 1.
7. the silver ion enrichment method according to claim 1, wherein the volume of the mixed solution II is smaller than the volume of the nitric acid solution II, and the volume ratio of the mixed solution II to the nitric acid solution II is 1: (3-5).
8. A quantitative kit for silver ions in a biological matrix is characterized by comprising a nitric acid solution I, a nitric acid solution II, a protein solution, a silver standard, a silver quality control product and an indium internal standard;
wherein the mass concentrations of the nitric acid solution I and the nitric acid solution II are respectively 0.5-2.0%; the concentration of the nitric acid solution II is higher than that of the nitric acid solution I; the protein solution is a fetal bovine serum albumin solution.
9. The silver ion quantification kit according to claim 8, wherein the silver ion quantification kit is detected by ICP-MS, and the silver standard comprises: 1) concentration of silver standard curve working solution: 300. 600, 1200, 2400, 4800, 7200, 10000 ng/mL-1The solvent is an indium-containing internal standard 1000 ng/mL-10.7% nitric acid; 2) silver standard urine sample concentration: 15. 30, 60, 120, 240, 360, 500 ng/mL-1The solvent is clinically collected urine of healthy subjects;
the silver quality control product comprises: 1) silver quality control working solution: 300. 700, 3000, 5000, 7600 ng.mL-1The solvent is an indium-containing internal standard 1000 ng/mL-10.7% nitric acid; 2) silver quality control urine sample: 15. 35, 150, 250, 380 ng/mL-1The solvent is urine of a healthy subject collected clinically.
10. The method for quantitatively detecting the silver ions in the biological matrix is characterized by comprising the steps of pretreating a biological matrix sample to obtain a sample to be detected and quantitatively detecting the sample to be detected by detection equipment, wherein the pretreatment comprises the following steps:
s01, adding a nitric acid solution I into the sample to enable the pH value of the system to be 6.5-8.0;
s02, adding a protein solution;
s03, adding a nitric acid solution II to enable the pH value of the system to be 1.0-3.0;
and S04, separating and transferring to obtain a sample to be detected.
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