CN115010940A - Aluminum-based metal organic framework material and preparation method and application thereof - Google Patents
Aluminum-based metal organic framework material and preparation method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
Abstract
The invention belongs to the field of advanced porous material technology application, and particularly relates to an aluminum-based metal organic framework material and a preparation method and application thereof. According to the invention, cheap aluminum chloride hexahydrate and 3, 5-pyrazole dicarboxylic acid are used as raw materials, and the hydrophilic aluminum-based metal organic framework material (Al-MOFs) can be prepared on a large scale, has good water stability and hydrophilic 1D pore channels, large specific surface area and proper pore size, and shows excellent circulation stability (10 times) and ultralow sensitivity (0.5 fmol/mu L) in the enrichment of a standard glycosylated peptide section sample. Further, the Al-MOFs material is successfully applied to the differential analysis of glycosylation peptide segments at different stages (normal, obesity, pre-diabetes and diabetes) in the occurrence and development process of diabetes. The material can be applied to the analysis and research of glycosylation proteomics in clinical large-scale samples.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to an aluminum-based metal organic framework material, a preparation method thereof and application of the aluminum-based metal organic framework material in enrichment of glycosylated peptide segments in the process of occurrence and development of diabetes.
Background
The glycosylation modification of the protein regulates various functions of the protein and participates in various complex physiological processes of an organism, and researches discover that the glycosylation modification of the protein is related to the occurrence and development of diabetes. Based on genome-wide association studies (GWAS), genes involved in the regulation of N-glycosylation have been shown to be associated with the development of diabetes. In addition, the complexity of the structure of plasma protein N-glycans has also been found to be associated with the risk of type 2 diabetes progression. For example, in the cohort of european and southeast asian ancestry, ST6 β -galactoside α -2, 6-sialyltransferase 1 (ST 6GAL 1) promotes α 2, 6-linked sialic acid to galactose-containing transfer substrates that have been shown to be associated with type 2 diabetes, confirming that highly branched N-glycans may be associated with the risk of diabetes. Conversely, protein glycosylation has also been found to play an important role in the prevention of diabetes. For example, glucose transporter 2 (glut-2) is a glucose sensor that regulates insulin secretion in beta cells and requires N-glycosylation to be retained on the beta cell surface to maintain glucose transport. Impaired glut-2 glycosylation results in decreased membrane glut-2 and insulin secretion, ultimately leading to diabetes. Based on previous research results, protein glycosylation may play different roles in promoting and preventing disease progression during the development of diabetes. In order to fully understand the effect of glycosylation on disease development, the modification of glycoprotein and the comprehensive analytical characterization of glycosylation sites are urgently needed to deeply understand the occurrence and development mechanism of diabetes and discover promising detection biomarkers. However, the direct use of mass spectrometry to identify site-specific glycan-modified glycoproteins in blood has presented a significant challenge in clinical application studies. There are several main reasons for this: (1) clinical samples are highly complex and diverse; (2) the dynamic range of the glycosylated protein is wide, and the fluctuation change is large; (3) compared with non-glycosylated peptide segments, the glycosylated peptide segments have extremely low abundance and are difficult to detect and identify. Therefore, enrichment and separation of the glycosylated peptide in the complex biological sample before mass spectrometry are the key for successfully analyzing the glycosylated protein and the key for analyzing the change of the glycosylated peptide in the process of generating and developing diseases.
Numerous research strategies have been proposed for the analytical study of glycosylated peptide fragments, in which hydrophilic interactions are favored for their unbiased enrichment capacity, retention of intact glycopeptide information, excellent biocompatibility and good reproducibility. Although these developed hydrophilic composites were used to capture glycopeptides and showed encouraging results, the synthesis of these composites generally required cumbersome functionalization steps, energy-intensive processes, and the use of expensive and environmentally unfriendly reagents. Not only greatly limits the scale production, but also restricts the subsequent commercial application. Therefore, the method further explores the hydrophilic material which is green and environment-friendly, has competitive price and is convenient for large-scale production, and is very important for the analysis and research of the glycosylated peptide in the process of disease occurrence and development.
Disclosure of Invention
The invention aims to provide an aluminum-based metal organic framework material with simple preparation method, good stability and good biocompatibility, a preparation method thereof and application of the material in enrichment of glycosylated peptide in the process of occurrence and development of diabetes.
The preparation method of the aluminum-based metal organic framework material provided by the invention comprises the following specific steps:
(1) dissolving sodium hydroxide and 3, 5-pyrazole dicarboxylic acid in a pure water solution to obtain a mixed solution; the weight ratio of the sodium hydroxide to the 3, 5-pyrazole dicarboxylic acid is as follows: 1: (1-3);
(2) reacting the mixed solution obtained in the step (1) for 1-3 hours under ultrasound to obtain a mixed solution;
(3) adding aluminum chloride hexahydrate into the mixed solution obtained in the step (2), and performing ultrasonic dispersion until the aluminum chloride hexahydrate is dissolved, wherein the concentration of the aluminum chloride hexahydrate is 1.5-6.0 mmol/L; reacting at 80-110 ℃ for 6-12h, and cooling to room temperature to obtain a reaction product; and then fully washing the reaction product by using deionized water to obtain a solid matter, performing vacuum drying for 10-24 hours at the temperature of 40-80 ℃ to obtain a product, namely the target aluminum-based metal organic framework material, which is marked as Al-MOFs.
In step (1) of the present invention, the weight ratio of sodium hydroxide to 3, 5-pyrazoledicarboxylic acid is preferably 1 (1 to 2.5), more preferably 1: 2.5.
In step (2) of the present invention, the ultrasonic reaction is preferably carried out for 1 to 2 hours, and more preferably for 2 hours.
In step (3) of the present invention, the concentration of the aluminum chloride hexahydrate solution is preferably 1.5 to 4.0 mmol/L, more preferably 4.0 mmol/L.
In step (3) of the present invention, the reaction temperature is preferably 80 to 100 ℃.
The aluminum-based metal organic framework material prepared by the invention has large specific surface area, good hydrophilic pore structure and stable biocompatibility, is easy to synthesize and prepare and is convenient for commercial popularization.
The aluminum-based metal organic framework material provided by the invention has high-efficiency separation effect, strong enrichment selectivity, excellent cycle stability (10 times) and ultralow sensitivity (0.5 fmol/mu L) on the glycosylated peptide, and can be used for selective separation, enrichment and mass spectrum identification of the glycosylated peptide in clinical complex samples.
Further, the method comprises the following specific steps: fully mixing the aluminum-based metal organic framework material with the glycosylated peptide solution of the sample, adding the mixture into 80-95% acetonitrile/0.1-1.0% trifluoroacetic acid buffer solution, uniformly dispersing, and incubating in an enzymolysis instrument at 30-40 ℃; centrifuging to separate material, washing with 80-95% acetonitrile/0.1-1.0% trifluoroacetic acid buffer solution for 3-5 times, and eluting with 25-35% acetonitrile/0.1-1.0% trifluoroacetic acid; spotting 0.5-1.5 μ L of eluate on MALDI-TOF-MS target plate, naturally drying, dripping 0.5-1.5 μ L of 2, 5-dihydroxybenzoic acid (DHB) solution with concentration of 15-25 mg/mL on the analyzed liquid drop to form thin layer matrix, drying, and performing mass spectrometry.
Specifically, the aluminum-based metal organic framework material prepared by the invention can be used for enrichment identification of glycopeptides in clinical serum samples (normal people, obese people, pre-diabetic people and diabetic people) and analysis of glycosylated proteomics. After the synthesized aluminum-based metal organic framework material is selectively enriched, mass spectrum high-flux detection is carried out, so that the signal intensity of the originally unidentified glycosylated peptide is greatly improved, and a clear signal peak of a glycosylated peptide section with high signal-to-noise ratio is obtained. The enrichment strategy is already practiced in multiple experiments such as standard HRP protein enzymatic hydrolysate, mixed solution of HRP protein enzymatic hydrolysate and Bovine Serum Albumin (BSA), HRP protein, human serum and the like, and can be used for disease differential typing through glycosylated protein difference in the development process of diabetes.
The invention has the beneficial effects that:
the synthesis method of the aluminum-based metal organic framework material is simple and convenient to operate, the raw materials are cheap and easy to obtain, and the large-scale production is easy to realize; the porous material has large specific surface area, proper hydrophilicity and pore-size structure, can successfully eliminate the interference of macromolecular protein and other impurities while separating and identifying glycopeptide, and achieves satisfactory effect in the application of clinical complex samples (in the development process of diabetes). Experimental results fully prove that the aluminum-based metal organic framework material prepared by the invention has excellent stability and biocompatibility and the capability of efficiently and selectively separating and identifying glycopeptides, and is suitable for separating and identifying glycopeptides in complex biological samples such as human serum, various tissue extracts, various cell proteins and the like. The aluminum-based metal organic framework material can be applied to analysis and identification of glycopeptides in serum/urine samples of patients with different types and periods of diseases in the future, so that potential biomarkers related to the diseases are found, and the aluminum-based metal organic framework material has the commercialization potential.
Drawings
FIG. 1 is a flow chart of the synthesis of an aluminum-based metal organic framework material.
FIG. 2 is a SEM photograph of Al-MOFs material in example 1.
FIG. 3 shows example 2 in which 10 -6 And (b) mass spectrograms of the HRP enzymatic hydrolysate (a) and (b) of the M after selective enrichment by using an aluminum-based metal organic framework material.
FIG. 4 shows the mass ratio of 1:500:500 mass spectrogram of HRP protein enzymolysis liquid, HRP protein and BSA protein mixed solution (a), (b) before selective enrichment and (c) and (d) after selective enrichment by aluminum-based metal organic framework material.
FIG. 5 shows the enrichment and separation of glycosylated peptide fragments in clinical serum samples (normal, obese, pre-diabetic, and diabetic) with the aluminum-based metal organic framework material in example 4. Wherein, (a) is based on PLS-DA model analysis, (b) is Venn diagram analysis, and (c) is heat map and trend analysis.
Detailed Description
Example 1: the process of the synthesis method of the aluminum-based metal organic framework material is shown in figure 1, and specifically comprises the following steps:
(1) dissolving 0.27 g of sodium hydroxide and 0.76 g of 3, 5-pyrazoledicarboxylic acid in a pure aqueous solution (75 mL) to obtain a mixed solution A;
(2) reacting the mixed solution A obtained in the step (1) for 2 hours under ultrasound, and recording the obtained mixed solution as B;
(3) adding 1.06 g of aluminum chloride hexahydrate into the mixed liquid B obtained in the step (2), performing ultrasonic dispersion until the aluminum chloride hexahydrate is dissolved, reacting for 10 hours at 90 ℃, cooling to room temperature to obtain a reaction product, fully washing with deionized water, and performing vacuum drying on the obtained solid matter at 50 ℃ for 18 hours to obtain a product C, namely the target aluminum-based metal organic framework material (Al-MOFs material).
The scanning electron micrograph of product C is shown in FIG. 2 (a, b).
Example 2: the aluminum-based metal organic framework material prepared in the example 1 is used for separation and enrichment and MALDI-TOF-MS detection of glycosylated peptide in standard HRP enzymatic hydrolysate.
(1) Preparation of standard HRP protein enzymatic hydrolysate: accurately weighing 1 mg of standard protein HRP, dissolving in 25 mM ammonium bicarbonate buffer solution, boiling at 100 ℃ for 10 minutes, cooling to room temperature, diluting to 1 mg/mL with 25 mM ammonium bicarbonate buffer solution, and then mixing the obtained product according to the mass ratio of the obtained product to the protein of 1: 40 adding proper amount of trypsin, and carrying out enzymolysis at 37 ℃ overnight for 18 hours.
(2) 10 mg of the aluminum-based metal organic framework material is washed by buffer solution containing 95% acetonitrile/0.1% trifluoroacetic acid for 3 times, and then dispersed in 1 mL of buffer solution containing 95% acetonitrile/0.1% trifluoroacetic acid, and the solution is dispersed to be uniform by ultrasonic wave to prepare 10 mg/mL solution.
(3) Enrichment of glycosylated peptides: adding 50 mu L of 95% acetonitrile/0.1% trifluoroacetic acid buffer solution into 50 mu L of the solution obtained in the step (2); then adding HRP standard protein enzymolysis liquid to make the peptide segment content be 10 -6 M, incubation reaction at 37 ℃ for 40 min; and (3) carrying out centrifugal separation to obtain the Al-MOFs material enriched with the glycopeptide, washing the Al-MOFs material with 200 mu L of 95% acetonitrile/0.1% trifluoroacetic acid buffer solution for three times to remove non-glycopeptide and other impurities, eluting the Al-MOFs material with 10 mu L of 30% acetonitrile/0.1% trifluoroacetic acid buffer solution for 30 minutes, and carrying out centrifugal separation to obtain an eluted peptide fragment solution.
(4) Target spotting: spotting 1 μ L of the eluate obtained in the step (3) on a MALDI-TOF-MS target plate, naturally drying in the air at room temperature, and dripping 1 μ L of 2, 5-dihydroxybenzoic acid solution (20 mg/mL of 50% ACN solution) as a matrix on the analyte droplets to generate a thin matrix layer, and drying; mass spectrometry was performed afterwards.
As can be seen in FIG. 3, prior to enrichment, 10 -6 Glycosylated peptide sections are difficult to identify in the HRP standard protein hydrolysate of M, a mass spectrogram mainly comprises non-glycopeptides (fig. 3 (a)), however, 23 glycosylated peptide peaks of HRP protein appear in the mass spectrogram after enrichment of aluminum-based metal organic framework materials, and signal peaks of the glycosylated peptide are remarkably improved (fig. 3 (b)).
Example 3: the aluminum-based metal organic framework material obtained in the example 1 is used for enrichment of HRP enzymatic hydrolysate, mixed solution of HRP protein and Bovine Serum Albumin (BSA) protein and MALDI-TOF-MS detection.
(1) According to the protein mass ratio of 1:500:500 mixing the HRP enzymatic hydrolysate with HRP and BSA protein solutions, adding 2 mu L of a standard mixed solution into 150 mu L of a 95% acetonitrile/0.1% trifluoroacetic acid buffer solution, adding 50 mu L of a dispersion solution (with the concentration of 10 mg/mL) of an aluminum-based metal organic framework material, and incubating for 40 minutes at 37 ℃; after centrifugation, the column was washed three times with 200. mu.L of 95% acetonitrile/0.1% trifluoroacetic acid buffer, and then eluted with 10. mu.L of 30% acetonitrile/0.1% trifluoroacetic acid buffer for 30 minutes, followed by centrifugation.
(2) Target spotting: and (3) spotting 1 mu L of the eluent obtained in the step (2) on a MALDI-TOF-MS target plate, placing the target plate in the air at room temperature for natural drying, taking 1 mu L of 2, 5-dihydroxybenzoic acid solution (20 mg/mL of 50% ACN solution) as a matrix, dripping the matrix on the analyte droplets to generate a thin matrix layer, and performing mass spectrometry after drying.
As can be seen from fig. 4, before enrichment, it is difficult to identify a glycosylated peptide fragment peak in a mixed solution of HRP enzymatic hydrolysate, HRP protein and BSA protein at a mass ratio of 1:500:500 (fig. 4 (a)); however, after the mixed solution is enriched by the aluminum-based metal organic framework material, a large number of glycosylated peptide peaks are clearly shown in the mass spectrum (fig. 4 (c)). In the mass spectrometry linear mode, HRP protein and BSA protein were identified in the supernatant before enrichment (fig. 4 (b)), while no signal peak for any protein was observed in the eluate after enrichment (fig. 4 (d)).
Example 4: the aluminum-based metal organic framework material obtained in the embodiment 1 is used for enrichment and nano-LC-MS/MS detection of glycosylated peptide in serum enzymolysis solution of clinical samples (normal people, obese people, pre-diabetic people and diabetic people).
(1) Preparation of clinical serum enzymolysis liquid: mu.L of human serum was diluted to 20. mu.L with deionized water and centrifuged at 14000 rmp for 15 minutes. The supernatant was collected, reacted at 60 ℃ for 30 minutes by adding 10 mM dithiothreitol, and then reacted at 37 ℃ for 60 minutes by adding 20 mM indoleacetic acid in the dark. After cooling, acetone is added according to the volume ratio of the protein to the acetone solution of 1:6, protein precipitation is carried out at-20 ℃, and the overnight reaction is carried out for 12 hours. Before proteolysis, diluting with 50 mM ammonium bicarbonate solution to obtain the final protein solubility of 2 mg/mL, and then, mixing the protein solution with the mixed solution according to the mass ratio of 1: 30 adding proper amount of trypsin into the supernatant, and carrying out enzymolysis for 16 hours at 37 ℃.
(2) Enrichment of glycosylated peptide fragments: taking 200 mu L of human serum enzymolysis liquid and 100 mu L of aluminum-based metal organic framework material dispersion liquid (the concentration is 10 mg/mL), adding the mixture into 150 mu L of 95% acetonitrile/0.1% trifluoroacetic acid buffer solution, and incubating and carrying out mixed rotation reaction for 40 minutes at 37 ℃; after centrifugation, the column was washed three times with 300. mu.L of 95% acetonitrile/1.0% trifluoroacetic acid buffer, and then eluted with 20. mu.L of 30% acetonitrile/0.1% trifluoroacetic acid buffer for 30 minutes, followed by centrifugation.
(3) Completely freeze-drying the eluted peptide fragment sample, dissolving the dry powder by using 8 mu L of chromatographic mobile phase A (0.1% formic acid aqueous solution), centrifuging for 15 minutes at 17000 rpm, and sucking the supernatant; mu.L of sample was injected, and elution analysis was performed by setting the chromatographic mobile phase B (0.1% formic acid in acetonitrile) for linear separation from 2% to 40% and then at a flow rate of 300 nL/min.
Enrichment is carried out by using an aluminum-based metal organic framework material, and 216 glycosylated peptide sections and 121 glycosylated proteins are identified in 1 mu L of serum. And the analysis of the differences in the identified glycosylated proteins between samples based on the PLS-DA model clearly distinguished normal, obese, pre-diabetic and diabetic serum samples (FIG. 5 a). As can be seen from the hierarchical clustering heatmap, the expression levels of the three groups of glycopeptides varied regularly as diabetes progressed. Compared with the normal group, the glycoprotein expression levels of the obesity group, the pre-diabetes group and the diabetes group were significantly higher (fig. 5 c). Secondly, there is a tendency to decrease with the development of diabetes (fig. 5 c). In addition, a sharp decrease in the number of glycoproteins was observed in the diabetic group (FIG. 5 c). The above results indicate that the identified differential glycopeptides and glycoproteins can be potential biomarkers for different stages of diabetes.
Example 5: the specific steps of the synthesis method of the aluminum-based metal organic framework material are the same as those of the embodiment 1, and the differences are as follows: in example 1 (3), the reaction time was changed to 5 hours.
Example 6: the aluminum-based metal organic framework material obtained in the embodiment 5 is used for enrichment separation and mass spectrum detection of glycopeptides in standard HRP enzymatic hydrolysate, and the specific steps of the enrichment and the detection are the same as those in (1), (2) and (3) in the embodiment 2.
After the aluminum-based metal organic framework material obtained in example 5 is enriched, only 16 glycopeptide signal peaks of HRP protein appear in a mass spectrogram, and the highest signal intensity is only 8000 more, which indicates that the reaction time has adverse effect on the enrichment effect.
Example 7: the specific steps of the synthesis method of the aluminum-based metal organic framework material are the same as those of the embodiment 1, and the differences are as follows: in example 1 (3), the feeding amount of aluminum chloride hexahydrate is reduced to 0.58 g.
Example 8: the aluminum-based metal organic framework material obtained in the example 7 is used for enrichment separation and mass spectrum detection of glycopeptides in standard HRP enzymatic hydrolysate, and the specific steps of the enrichment and the detection are the same as those in (1), (2) and (3) in the example 2.
After the enrichment of the aluminum-based metal organic framework material obtained in example 7, only 18 glycopeptide signal peaks of HRP protein appear in the mass spectrogram, and the highest signal intensity is only 9000 more, which indicates that the formation of the pore diameter of the MOFs material has a certain influence on the enrichment effect.
Example 9: the aluminum-based metal organic framework material obtained in the example 1 is used for enrichment separation and mass spectrometry detection of glycopeptides in standard HRP enzymatic hydrolysate, the specific steps of enrichment and detection are the same as (1), (2) and (3) in the example 2, and only the 30% acetonitrile/0.1% trifluoroacetic acid buffer solution of (2) in the example 2 is changed into 50% acetonitrile/0.1% trifluoroacetic acid buffer solution.
Example 9 enrichment of glycopeptides under the conditions, only 14 glycopeptide signal peaks of HRP protein appeared in the mass spectrum, indicating ACN/H 2 The concentration of the O elution solution also has an effect on the enrichment effect.
Claims (9)
1. The preparation method of the aluminum-based metal organic framework material is characterized by comprising the following specific steps:
(1) dissolving sodium hydroxide and 3, 5-pyrazole dicarboxylic acid in a pure water solution to obtain a mixed solution; the weight ratio of the sodium hydroxide to the 3, 5-pyrazole dicarboxylic acid is as follows: 1: (1-3);
(2) reacting the mixed solution obtained in the step (1) for 1-3 hours under ultrasound to obtain a mixed solution;
(3) adding aluminum chloride hexahydrate into the mixed solution obtained in the step (2), and performing ultrasonic dispersion until the aluminum chloride hexahydrate is dissolved, wherein the concentration of the aluminum chloride hexahydrate is 1.5-6.0 mmol/L; reacting at 80-110 ℃ for 6-12h, and cooling to room temperature to obtain a reaction product; and then fully washing the reaction product by using deionized water to obtain a solid matter, performing vacuum drying for 10-24 hours at the temperature of 40-80 ℃ to obtain a product, namely the target aluminum-based metal organic framework material, which is marked as Al-MOFs.
2. The method for preparing an aluminum-based metal organic framework material according to claim 1, wherein the weight ratio of the sodium hydroxide to the 3, 5-pyrazole dicarboxylic acid in the step (1) is 1 (1-2.5).
3. The method for preparing an aluminum-based metal organic framework material according to claim 1, wherein the ultrasonic reaction in step (2) is carried out for 1-2 h.
4. The method for preparing an aluminum-based metal-organic framework material according to claim 1, wherein the concentration of the aluminum chloride hexahydrate in step (3) is 1.5-4.0 mmol/L.
5. The method for preparing an aluminum-based metal organic framework material according to claim 1, wherein the reaction temperature in step (3) is 80-100 ℃.
6. An aluminum-based metal organic framework material obtainable by the preparation process as claimed in any one of claims 1 to 5.
7. Use of the aluminum-based metal organic framework material of claim 6 for selective separation, enrichment and mass spectrometric identification of glycosylated peptides in complex samples.
8. The application of claim 7, comprising the following steps:
fully mixing the aluminum-based metal organic framework material with the glycosylated peptide solution of the sample, adding the mixture into 80-95% acetonitrile/0.1-1.0% trifluoroacetic acid buffer solution, uniformly dispersing, and incubating in an enzymolysis instrument at 30-40 ℃;
centrifuging to separate material, washing with 80-95% acetonitrile/0.1-1.0% trifluoroacetic acid buffer solution for 3-5 times, and eluting with 25-35% acetonitrile/0.1-1.0% trifluoroacetic acid;
spotting 0.5-1.5 μ L of the eluate on a MALDI-TOF-MS target plate, naturally drying, dripping 0.5-1.5 μ L of 2, 5-dihydroxybenzoic acid (DHB) solution with concentration of 15-25 mg/mL on the analyte droplets to form a thin-layer matrix, drying, and performing mass spectrometry.
9. Use according to claim 7 or 8, wherein the sample is a serum sample.
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