Mouse cerebrospinal fluid in-situ extraction and detection method based on nano-spray ionization mass spectrometry

Abstract

The invention discloses a mouse cerebrospinal fluid in-situ extraction and detection method based on nano-spray ionization mass spectrometry, and belongs to the field of detection. The method specifically comprises the following four steps: etching a micro-extraction probe, performing electrochemical synthesis on an extraction material on the surface of the probe, performing in-situ enrichment on mouse cerebrospinal fluid, performing desorption and nano-spray ionization mass spectrometry analysis, and performing data analysis. The method is high in universality, high in sensitivity and simple in device. Wherein the microextraction probe specifically enriches a target object in cerebrospinal fluid, and matrix interference is reduced. Rapid, high-flux and high-sensitivity detection of trace target objects is realized by the nano-spray ionization mass spectrometry. The in-situ extraction and detection method for the mouse cerebrospinal fluid based on the nano-spray ionization mass spectrometry is green, efficient and large in application potential, and can meet the actual requirements of rapid in-situ extraction and detection of substances in the mouse cerebrospinal fluid. The adsorption material used in the method can be synthesized according to the type of the target object, and the technology has important significance for improving the analysis efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biological sample detection, and in particular to a method for preparing a microprobe for online extraction of cerebrospinal fluid and a method for detecting small molecules in mouse cerebrospinal fluid based on a microprobe and nanospray mass spectrometry. Technical Background

[0002] Cerebrospinal fluid is an important window that reflects changes in the brain environment and is usually used as an important physiological matrix for studying substances in the brain. Small molecules in cerebrospinal fluid play an important role in maintaining the physiological functions of cerebrospinal fluid, participating in neural signal transduction, neurotransmitter metabolism, and blood-cerebrospinal fluid barrier function. By detecting these small molecules, important information can be provided for the diagnosis and treatment of neurological diseases. In recent years, with the deepening of brain chemistry research, the demand for cerebrospinal fluid testing has also increased. Due to the small sample volume of cerebrospinal fluid (in the commonly used mouse model, if the mouse is to survive, the safe amount of cerebrospinal fluid for a single mouse is about 3μL), complex composition, and serious matrix interference, it is very difficult to detect specific targets in cerebrospinal fluid, and the sensitivity, analysis timeliness, analysis throughput, and qualitative and quantitative capabilities of the detection method are extremely high.

[0003] Among the existing cerebrospinal fluid detection technologies, the methods of cerebrospinal fluid detection mainly include microelectrode method and microdialysis tandem chromatography-mass spectrometry. The microelectrode method has been used for in situ real-time brain chemistry research in mice. It is often used to detect pH, specific ions and other electroactive substances, and it is difficult to conduct a comprehensive chemical composition analysis (JACS Au., 2023, 3, 1572-1582; ​​Anal. Chim. Acta., 2022, 1191, 339209). Microdialysis technology is a common technology for cerebrospinal fluid sampling, using a specific dialysis membrane to achieve continuous sampling of the target (Eur. J. Pharm. Biopharm., 2016, 104, 117-130; Patent CN202110525342). However, microdialysis collection is difficult to achieve specific collection, and a large amount of matrix interference will occur. Therefore, in subsequent testing, separation detection technologies such as chromatography-mass spectrometry are usually still required (J. Pharm. Biomed. Anal., 2020, 179, 112996; Food Funct., 2023, 14, 8893). The sampling technology is demanding and the analysis is time-consuming, and the timeliness of the analysis of highly active small molecules is also insufficient. Micropipette is also an efficient method for sampling trace cerebrospinal fluid (patent CN200710179047). However, how to avoid the high sample volume requirements, limited detection sensitivity and insufficient timeliness of separation detection technologies such as chromatography-mass spectrometry on the basis of efficient sampling, and achieve rapid and efficient detection of small molecules in cerebrospinal fluid, is still a direction that urgently needs development.

[0004] Nano-electrospray direct mass spectrometry detection technology based on micro-nano spray needles has fast analysis and high analysis throughput (Se Pu., 2021, 39, 1140-1150; J. Food Drug Anal., 2021, 29, 468-489), and is compatible with micropipette cerebrospinal fluid sampling technology, and is expected to become a new technology for trace cerebrospinal fluid detection. However, high concentrations of non-volatile salts and other matrices in cerebrospinal fluid can inhibit the signal of low-content target substances, making detection difficult. Summary of the invention

[0005] In order to make up for the problems of high sample quantity requirement, limited detection sensitivity and timeliness in the existing cerebrospinal fluid detection technology, the present invention introduces a microextraction probe on the basis of micropipette sampling, and provides a method for detecting small molecules in mouse cerebrospinal fluid based on microextraction probe and nanospray mass spectrometry, so as to realize rapid and highly sensitive analysis of target objects in cerebrospinal fluid.

[0006] The invented analytical method comprises four steps: etching of the microextraction probe and electrochemical synthesis of the extraction material on the probe surface, in situ enrichment of mouse cerebrospinal fluid, desorption and nanospray mass spectrometry analysis, and data analysis. The specific steps are as follows:

[0007] Step 1, etching of the microextraction probe and electrochemical synthesis of the extraction material on the probe surface: placing a stainless steel needle in an acidic etching solution, heating and etching, and then immersing the etched probe in an electrodeposition solution, and synthesizing the extraction material at the tip of the etched stainless steel needle by electrochemistry to obtain a microextraction probe; the electrodeposition solution is composed of a polymer monomer and a dopant, the polymer monomer is one or more of pyrrole, vinylpyrrolidone, and aniline, and the dopant is one or more of sulfuric acid, trifluoroacetic acid, nitric acid, and phosphoric acid;

[0008] Step 2, inserting a micropipette into the cisterna magna of the mouse, and when cerebrospinal fluid seeps into the pipette, inserting the microextraction probe prepared in step 1 into the cerebrospinal fluid micropipette for in situ extraction, and after a period of time, taking out the microextraction probe and cleaning it;

[0009] Step 3, inserting the microextraction probe after extracting cerebrospinal fluid in step 2 into a nanospray needle pre-added with solvent for desorption, then placing the nanospray needle in front of the mass spectrometer inlet, applying voltage to the nanospray needle, setting a mass spectrometer acquisition method, and performing mass spectrometer detection;

[0010] Step 4: mass spectrometry data analysis.

[0011] Polypyrrole was in situ grown on the probe by electrochemical synthesis, and specific extraction of polyphenols was achieved through hydrogen bonding, П-Π interaction and electrostatic interaction between polypyrrole and polyphenols.

[0012] The prepared polypyrrole solid phase microextraction probe can efficiently and specifically extract polyphenols in mouse cerebrospinal fluid, avoiding the chromatographic separation step, and realizing simultaneous qualitative and quantitative analysis of multiple targets in trace cerebrospinal fluid, with efficient and rapid detection.

[0013] In one embodiment, the outer diameter of the stainless steel needle in step 1 is 0.1 to 0.5 mm and the length is 20 to 50 mm;

[0014] The etching solution is a 5% to 15% hydrochloric acid solution, the etching temperature is 50 to 90° C., and the etching time is 1.5 to 2.5 hours.

[0015] In one embodiment, 0.1-0.5 mol / L pyrrole and 0.1-1.0 mol / L sulfuric acid aqueous solution are used as the electrodeposition solution.

[0016] In one embodiment, the electrochemical synthesis mode can be selected from voltammetry, chronoamperometry, chronopotentiometry, and chronocoulometry.

[0017] In one embodiment, the chronopotentiometry method is used, the potential can be selected from 1.1 to 1.5 V, and the electrodeposition time can be selected from 30 to 180 s.

[0018] In one embodiment, the micropipette in step 2 is drawn from a quartz glass tube, and the tip size of the micropipette is 1 to 2 μm;

[0019] The extraction time in step 2 can be selected from 1 to 15 minutes;

[0020] The cleaning solvent is water.

[0021] In one embodiment, the nanospray needle in step 3 is drawn from a quartz glass tube, and the tip size of the obtained micro glass pipette is about 1 to 2 μm; the pre-added solvent is a pure solution or a mixed solution of methanol, acetonitrile, ethanol, isopropanol, acetone, or water, and the added volume is 0.5 μL to 10 μL; and the desorption time is 3 to 10 min.

[0022] In one embodiment, the method of applying voltage to the spray needle is to retain the extraction probe inserted into the spray needle and apply voltage to the microextraction probe, or to insert a copper wire into the microextraction probe and apply voltage to the copper wire, or to deposit a metal coating such as gold, silver, platinum, etc. on the outer wall of the nanospray spray needle and apply voltage to the outer wall of the spray needle, and the voltage value is 1.5 to 3.5 kV.

[0023] In one embodiment, the mass spectrometry acquisition method includes positive ion mode, negative ion mode, full scan mode, selected ion monitoring mode, and selected reaction monitoring mode.

[0024] In one embodiment, the order of sample detection is that the standards are detected from low to high concentrations (c1, c2, ..., cn), and then the samples are detected (x1, x2, ..., xm).

[0025] In one embodiment, each sample is tested no less than 3 times.

[0026] In one embodiment, the data processing in step 4 uses the signal peak area intensity of the target ion in the mass spectrum from the start of spray detection to the disappearance of the signal for quantitative analysis, and the data analysis specifically includes:

[0027] Step 4-1, extracting the sample quantitative ion peak areas (Ac1, Ac2, ..., Acn; Ax1, Ax2, ..., Axm) from the obtained mass spectrum respectively;

[0028] Step 4-2, with c1, c2, ..., cn as the horizontal coordinate values ​​and Ac1, Ac2, ..., Acn as the vertical coordinate values, draw a graph, perform linear fitting on the data points in the graph, and obtain a linear equation;

[0029] Step 4-3, substitute Ax1, Ax2, ..., Axm into the linear equation to calculate the target concentration x1, x2, ..., xm in the sample to be tested.

[0030] Beneficial effects:

[0031] (1) A highly sensitive analytical method has been established for trace CSF samples, which does not require a large sample volume and is conducive to continuous CSF measurement in living subjects;

[0032] (2) A microextraction probe was designed to achieve specific enrichment of the target and avoid interference from the complex matrix of cerebrospinal fluid;

[0033] (3) The microextraction probe is combined with nanospray mass spectrometry, which eliminates the need for chromatographic separation steps and enables simultaneous qualitative and quantitative analysis of multiple targets in trace amounts of cerebrospinal fluid, making the detection efficient and rapid. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the in-situ extraction and detection method of mouse cerebrospinal fluid by nanospray mass spectrometry of the present invention;

[0035] Figure 2 This is a scanning electron microscope image of the polypyrrole microextraction probe of Example 1;

[0036] Figure 3 Schematic diagram of the nanospray mass spectrometer of Example 1;

[0037] Figure 4 This is the ion flow diagram of the detection of 6 polyphenols in Example 1;

[0038] Figure 5 is the mass spectrum of the six polyphenols in Example 1;

[0039] Figure 6 Graphs showing the standard curves of the six polyphenols in Example 1; A is the standard curve of 3-hydroxyphenylacetic acid, homovanillic acid (black legend, left y-axis), caffeic acid and ferulic acid (grey legend, right y-axis); B is the standard curve of epicatechin (grey legend) and epigallocatechin gallate (black legend);

[0040] Figure 7 The figure shows the dynamic changes of the concentrations of the six polyphenols in Example 1 in the cerebrospinal fluid. DETAILED DESCRIPTION

[0041] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0042] The technical solution of the present invention is further described below through embodiments in conjunction with the accompanying drawings, but the protection scope of the present application is not limited by the specific conditions of these embodiments.

[0043] Embodiment 1:

[0044] The in situ extraction and detection method of mouse cerebrospinal fluid based on nanospray mass spectrometry of the present invention was used to determine six polyphenols in cerebrospinal fluid. The specific steps are as follows:

[0045] (1) A stainless steel acupuncture needle with an outer diameter of 120 μm was used as the substrate of the microextraction probe. A 9% hydrochloric acid solution was prepared in a 100 ml beaker. The stainless steel needle tip was immersed in the hydrochloric acid solution for 4 cm and placed in a 70°C oven for 2 h. The needle was washed with water twice and with anhydrous ethanol once. The etched stainless steel needle was observed under an optical microscope and the needle tip of 20 to 40 μm was taken for use.

[0046] An aqueous solution containing 0.35 mol / L pyrrole and 0.5 mol / L sulfuric acid was used as the electrodeposition solution, and the electrodeposition was performed using a CHI800D electrochemical analyzer. The specific electrodeposition conditions were as follows: three-electrode system, potential 1.2 V, time 60 s, hysteresis 0 s. A polypyrrole probe with an outer diameter of 40 to 70 μm was selected for use. The scanning electron microscope image of the prepared polypyrrole microextraction probe is shown in Figure 2 .

[0047] (2) A quartz glass tube with an outer diameter of 1.5 mm and an inner diameter of 0.86 mm was pulled apart using a P-2000 / G laser microelectrode pulling apparatus under the conditions of HEAT: 450, FIL: 4, VEL: 50, DEL: 145, and PUL: 0 to obtain two micro glass pipettes. The tip size of the prepared micro glass pipettes was about 1 to 2 μm.

[0048] After 2 days of adaptive feeding, 2-month-old C57BL / 6J male mice were randomly divided into experimental groups (1h group, 2h group, 4h group, 6h group and 8h group) and control groups, with 3 mice in each group. The experimental group was gavaged with 0.3ml polyphenol coffee solution, and the control group was gavaged with 0.3ml distilled water. After gavage, cerebrospinal fluid of mice was collected with a micro-glass pipette at 1h, 2h, 4h, 6h and 8h, respectively, and cerebrospinal fluid of the control group was collected 1h after gavage.

[0049] When the mouse cerebrospinal fluid was drawn with a micropipette, the polypyrrole microextraction probe was immediately inserted into the microtube for enrichment for 10 minutes. After enrichment, the polypyrrole microprobe was taken out and washed once in distilled water to remove impurities such as soluble salts and reduce interference with mass spectrometry signals.

[0050] (3) Use a micro-glass pipette to absorb 1 mg of 1 ppm captopril ethanol solution, insert the washed polypyrrole microextraction probe into the micro-glass tube for desorption for 4 minutes, then remove the probe. Then place the micro-glass pipette on a movable platform in front of the mass spectrometer entrance, and adjust the position so that the horizontal distance between the tip of the glass pipette and the mass spectrometer entrance is 6 mm and the vertical distance is 3 mm. Insert one end of the copper wire into the micro-glass tube to 2 mm below the liquid surface, and connect the other end to the high voltage output port of the mass spectrometer ( Figure 3 ).

[0051] The sample was determined in MRM mode, and seven substances (3-hydroxyphenylacetic acid, homovanillic acid, caffeic acid, ferulic acid, epicatechin, epigallocatechin gallate and captopril) were detected simultaneously. The quantitative / qualitative ion pairs and fragmentation energy were listed in Table 1. Captopril was used as an internal standard.

[0052] Table 1 MRM detection conditions for 7 targets

[0053]

[0054]

[0055] (4) Mass spectrometry data: In the obtained ion flow diagram ( Figure 4 ), based on the quantitative ion pair signals of six polyphenols ( Figure 5 ) to obtain the peak area, and the standard curves of the six polyphenols were obtained with the peak area of ​​the quantitative ion pair of the six polyphenol standards / the peak area of ​​the captopril quantitative ion as the ordinate and the concentration as the abscissa ( Figure 6): The linear range of 3-hydroxyphenylacetic acid is 0.5-20 μg / mL (Y=0.2365X+0.0668, R2=0.997), i.e., the absolute amount is 0.25-10 ng, and the detection limit is 0.13 μg / mL (0.06 ng); the linear range of homovanillic acid is 1-20 μg / mL (Y=0.1087X-0.0372, R2=0.996), i.e., the absolute amount is 0.5-10 ng, and the detection limit is 0.39 μg / mL (0.20 ng); the linear range of caffeic acid is 0.1-20 μg / mL (Y=1.050X+0.188, R2=0.997), i.e., the absolute amount is 0.05-10 ng, and the detection limit is 0.031 μg / mL (0.016 ng); ferulic acid The linear range of is 0.1-20 μg / mL (Y=1.325X+0.292, R2=0.999), i.e., the absolute amount is 0.05-10 ng, and the detection limit is 0.027 μg / mL (0.014 ng); the linear range of epicatechin is 1-20 μg / mL (Y=0.006979X+0.00542, R2=0.994), i.e., the absolute amount is 0.5-10 ng, and the detection limit is 0.35 μg / mL (0.18 ng); the linear range of epigallocatechin gallate is 1-20 μg / mL (Y=0.007013X+0.00411, R2=0.997), i.e., the absolute amount is 0.5-10 ng, and the detection limit is 0.28 μg / mL (0.14 ng).

[0056] Then, the quantitative ion peak area of ​​the mouse cerebrospinal fluid sample to be tested / captopril quantitative ion peak area is brought into the standard curve to obtain the concentration of the test substance, and then a graph of the change of polyphenols in the mouse cerebrospinal fluid over time is drawn ( Figure 7 ).

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry, characterized in that: The method comprises the following steps: Step 1, etching of the microextraction probe and electrochemical synthesis of the extraction material on the probe surface: placing a stainless steel needle in an acidic etching solution, heating and etching, and then immersing the etched probe in an electrodeposition solution, and synthesizing the extraction material at the tip of the etched stainless steel needle by electrochemistry to obtain a microextraction probe; the electrodeposition solution is composed of a polymer monomer and a dopant, the polymer monomer is one or more of pyrrole, vinylpyrrolidone, and aniline, and the dopant is one or more of sulfuric acid, trifluoroacetic acid, nitric acid, and phosphoric acid; Step 2, inserting a micropipette into the cisterna magna of the mouse, and when cerebrospinal fluid seeps into the pipette, inserting the microextraction probe prepared in step 1 into the cerebrospinal fluid micropipette for in situ extraction, and after a period of time, taking out the microextraction probe and cleaning it; Step 3, inserting the microextraction probe after extracting cerebrospinal fluid in step 2 into a nanospray needle pre-added with solvent for desorption, then placing the nanospray needle in front of the mass spectrometer inlet, applying voltage to the nanospray needle, setting a mass spectrometer acquisition method, and performing mass spectrometer detection; Step 4: mass spectrometry data analysis.

2. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 1, characterized in that: In step 1, the outer diameter of the stainless steel needle is 0.1 to 0.5 mm, and the length is 20 to 50 mm; The etching solution is a 5% to 15% hydrochloric acid solution, the etching temperature is 50 to 90° C., and the etching time is 1.5 to 2.5 hours.

3. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 1, characterized in that: A 0.1-0.5 mol / L pyrrole and a 0.1-1.0 mol / L sulfuric acid aqueous solution are used as the electrodeposition solution.

4. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 1, characterized in that: The electrochemical synthesis mode can be selected from voltammetry, chronoamperometry, chronopotentiometry and chronocoulometry.

5. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 4, characterized in that: The chronopotentiometry method is adopted, the potential can be selected from 1.1 to 1.5 V, and the electrodeposition time can be selected from 30 to 180 s.

6. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 9, characterized in that: In step 2, the micropipette is drawn from a quartz glass tube, and the tip size of the micropipette is 1 to 2 μm; The extraction time in step 2 can be selected from 1 to 15 minutes; The cleaning solvent is water.

7. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 1, characterized in that: In step 3, the nanospray needle is drawn from a quartz glass tube, and the tip size of the obtained micro glass pipette is about 1 to 2 μm; the pre-added solvent is a pure solution or a mixed solution of methanol, acetonitrile, ethanol, isopropanol, acetone, or water, and the added volume is 0.5 μL to 10 μL; and the desorption time is 3 to 10 min.

8. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 1, characterized in that: The method of applying voltage to the spray needle is to keep the extraction probe inserted into the spray needle and apply voltage to the microextraction probe, or to insert a copper wire into the microextraction probe and apply voltage to the copper wire, or to deposit a metal coating such as gold, silver, platinum, etc. on the outer wall of the nanospray spray needle and apply voltage to the outer wall of the spray needle, and the voltage value is 1.5 to 3.5 kV.

9. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 1, characterized in that: Mass spectrometry acquisition methods include positive ion mode, negative ion mode, full scan mode, selected ion monitoring mode, and selected reaction monitoring mode.

10. The method for in situ extraction and detection of mouse cerebrospinal fluid based on nanospray mass spectrometry according to claim 1, characterized in that: In step 4, the data processing uses the signal peak area intensity of the target ion in the mass spectrum from the start of spray detection to the disappearance of the signal for quantitative analysis; the data analysis specifically includes: Step 4-1, extracting the sample quantitative ion peak areas (Ac1, Ac2, ..., Acn; Ax1, Ax2, ..., Axm) from the obtained mass spectrum respectively; Step 4-2, with c1, c2, ..., cn as the horizontal coordinate values ​​and Ac1, Ac2, ..., Acn as the vertical coordinate values, draw a graph, perform linear fitting on the data points in the graph, and obtain a linear equation; Step 4-3, substitute Ax1, Ax2, ..., Axm into the linear equation to calculate the target concentration x1, x2, ..., xm in the sample to be tested.

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