Sample pretreatment method, method for detecting ratio of 5-methylcytosine to cytosine, and kit

Abstract

The invention relates to the technical field of biochemical detection, in particular to a sample pretreatment method, a method for detecting the ratio of 5-methylcytosine to cytosine and a kit. The sample pretreatment method comprises the following steps: providing a sample containing substances to be detected, wherein the substances to be detected comprise 5-methylcytosine and cytosine; the method comprises the following steps: mixing magnetic beads containing sulfonic acid groups with a sample, carrying out adsorption treatment, and then carrying out leaching treatment by using an alcoholic solution to obtain a to-be-detected substance-magnetic bead compound; eluting the to-be-detected substance-magnetic bead compound by using the eluent to obtain a to-be-detected sample; the eluent comprises at least one of a formic acid-alcohol solution and an ammonia water-alcohol solution. The sample pretreatment method is simple in process and low in cost, the to-be-detected sample containing the 5-methylcytosine and the cytosine can be efficiently obtained, the ratio of the 5-methylcytosine to the cytosine can be accurately quantified in a high-sensitivity mode on the basis of the electrospray high-efficiency ion mobility spectrometry technology subsequently, and the method has good application prospects.

Description

Technical Field

[0001] This application belongs to the field of biochemical detection technology, and in particular relates to a sample pretreatment method, a method for detecting the ratio of 5-methylcytosine to cytosine, and a reagent kit. Background Technology

[0002] DNA methylation is an epigenetic modification that involves the covalent attachment of methyl groups to DNA molecules. This process can occur in cytosine (C), adenine (A), or guanine (G). As an important epigenetic modification in eukaryotes, the abnormal expression of DNA methylation is closely related to the occurrence and development of various diseases, including cancer, diabetes, neurological disorders, and autoimmune diseases.

[0003] 5-methylcytosine (5-mC) is one of the most important epigenetic modifications in DNA, typically occurring at the 5th carbon atom of cytosine in the CpG dinucleotide sequence. As a normal component of DNA, 5-mC accounts for only about 3% of all cytosine, and approximately 2.6–4.8% in human DNA. As a key regulator of gene expression, 5-mC plays a crucial role in maintaining chromatin structure, ensuring DNA stability, and facilitating protein-protein interactions. Its global-level detection is of great significance for understanding cellular epigenetic status, identifying disease biomarkers, and assessing the impact of the environment on the epigenome.

[0004] Currently, commonly used methods for detecting overall genome methylation levels include high-performance capillary electrophoresis (HPCE), high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), and enzyme-linked immunosorbent assay (ELISA). While these methods can quantify the average DNA methylation level at all CpG sites in the target genome, they still have certain limitations. For example, HPCE has low sensitivity, long analysis time, and poor stability; HPLC has a cumbersome procedure and long analysis time; LC-MS / MS instruments are expensive, complex to operate, long analysis time, require high maintenance, and need to be operated by professional personnel; ELISA is prone to cross-reactivity, has strong antibody dependence, insufficient specificity, and is difficult to develop.

[0005] Overall, existing testing methods struggle to balance speed, sensitivity, and cost-effectiveness, making it difficult to meet the needs of rapid clinical testing. Summary of the Invention

[0006] The purpose of this application is to provide a sample pretreatment method, a method for detecting the ratio of 5-methylcytosine to cytosine, and a kit, aiming to solve how to pretreat samples containing 5-methylcytosine and cytosine to accurately quantify the ratio of 5-methylcytosine to cytosine in a low-cost, efficient, and highly sensitive manner.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0008] In a first aspect, this application provides a sample pretreatment method, comprising: Provide a sample containing an analyte, said analyte including 5-methylcytosine and cytosine; The sample was mixed with magnetic beads containing sulfonic acid groups for adsorption treatment, and then washed with an alcohol solution to obtain the analyte-magnetic bead complex. The analyte-magnetic bead complex is eluted with an eluent to obtain the test sample; the eluent includes at least one of formic acid-alcohol solution and ammonia-alcohol solution.

[0009] In some embodiments, the magnetic bead comprises a polystyrene-divinylbenzene copolymer with a magnetic core coated on the surface of the magnetic core and bonded with sulfonic acid groups; And / or, the magnetic beads comprise a hybrid strong cation exchange adsorbent with a magnetic core coated on the surface of the magnetic core, the hybrid strong cation exchange adsorbent comprising benzenesulfonic acid groups and hydrophobic groups; And / or, the average particle size of the magnetic beads is 1~15μm.

[0010] In some embodiments, the alcohol solution comprises a methanol solution with a volume percentage of 5% to 10%; And / or, the formic acid-alcohol solution in the eluent comprises a formic acid-methanol solution with a volume percentage of 0.00001% to 1%; And / or, the ammonia-alcohol solution in the eluent comprises an ammonia-methanol solution with a volume percentage of 0.001% to 1%.

[0011] In some embodiments, the rinsing treatment with alcohol solution includes: first rinsing with a methanol solution of 5% to 7% by volume for 1 min to 2 min, and then rinsing with a methanol solution of 7% to 10% by volume for 1 min to 2 min. And / or, the elution treatment of the analyte-magnetic bead complex with the elution buffer includes: eluting with an ammonia-methanol solution of 0.001%~0.1% by volume for 1 min~2 min.

[0012] In some embodiments, the magnetic beads are activated before being mixed with the sample, and then water is added for equilibration. And / or, the magnetic beads are mixed with the sample in a formic acid solution with a volume percentage of 0.01% to 1% formic acid for the adsorption treatment.

[0013] In some embodiments, the analyte is derived from free bases released after DNA degradation; And / or, the sample containing the analyte includes a standard curve sample, a quality control sample, and an analytical sample arranged side by side.

[0014] Secondly, this application provides a method for detecting the ratio of 5-methylcytosine to cytosine, comprising: The sample containing the analyte is processed using the sample pretreatment method provided in the first aspect of this application to obtain the sample to be tested. The sample was analyzed by ion mobility spectrometry to obtain the ratio of 5-methylcytosine to cytosine.

[0015] In some embodiments, the ion mobility spectrometry includes electrospray ionization source ion mobility spectrometry, and the detection conditions include: Positive ion mode, ion source voltage 1800~2100V, migration tube voltage 8000~10000V, inlet temperature 170~240℃, migration tube temperature 170~240℃, ion gate voltage 50~60V, ion gate voltage pulse width 80~120μs, migration gas and exhaust gas flow rates 1.0~1.5 L / min; And / or, the detection method has a lower limit of quantification of 1% for the 5-methylcytosine to cytosine ratio.

[0016] Thirdly, this application provides a kit for pretreatment of samples containing 5-methylcytosine and cytosine, comprising: Magnetic beads: contain sulfonic acid groups on their surface; Rinse solution: alcohol solution; Eluent: at least one of formic acid-alcohol solution and ammonia-alcohol solution.

[0017] In some embodiments, the magnetic bead comprises a polystyrene-divinylbenzene copolymer with a magnetic core coated on the surface of the magnetic core and bonded with sulfonic acid groups; And / or, the magnetic beads comprise a hybrid strong cation exchange adsorbent with a magnetic core coated on the surface of the magnetic core, the hybrid strong cation exchange adsorbent comprising benzenesulfonic acid groups and hydrophobic groups; And / or, the average particle size of the magnetic beads is 1~15μm.

[0018] In some embodiments, the alcohol solution comprises a methanol solution with a volume percentage of 5% to 10%; And / or, the formic acid-alcohol solution in the eluent comprises a formic acid-methanol solution with a volume percentage of 0.00001% to 1%; And / or, the ammonia-alcohol solution in the eluent comprises an ammonia-methanol solution with a volume percentage of 0.001% to 1%.

[0019] In some embodiments, the kit further includes formic acid solution with a volume percentage of 0.01% to 1% for diluting the magnetic beads and the sample for adsorption; And / or, the kit may further include an alcohol solution for dispersing and activating the magnetic beads.

[0020] The sample pretreatment method provided in the first aspect of this application involves adding unique magnetic beads with sulfonic acid groups on their surface to the sample for adsorption extraction. Because the strongly acidic sulfonic acid groups on the surface of the magnetic beads have a strong cation exchange effect, the analytes, namely 5-methylcytosine and cytosine (which generally exist in cationic form under adsorption conditions), can be adsorbed in the sample based on ionic bonds. Thus, 5-methylcytosine and cytosine can be easily bound to the magnetic beads. Then, the analyte-magnetic bead complex is obtained by elution with an alcohol solution. Subsequently, at least one of formic acid-alcohol solution and ammonia-alcohol solution is used as the eluent to change the interaction force between the analyte and the magnetic beads, thereby causing the analyte to detach from the magnetic beads, resulting in a sample containing 5-methylcytosine and cytosine. This sample pretreatment method is not only simple and low-cost, but also can efficiently obtain high-purity test samples containing 5-methylcytosine and cytosine. It can then be used for highly sensitive and accurate quantification of the 5-methylcytosine to cytosine ratio using ion mobility spectrometry. Therefore, it has great application prospects in the detection of the 5-methylcytosine to cytosine ratio, especially in the field of DNA methylation detection.

[0021] The method for detecting the 5-methylcytosine to cytosine ratio provided in the second aspect of this application utilizes the sample pretreatment method provided in the first aspect of this application to treat a sample containing 5-methylcytosine and cytosine, obtaining a test sample. Then, ion mobility spectrometry is used to detect the 5-methylcytosine to cytosine ratio in the test sample. This detection method allows for low-cost, efficient, and highly sensitive precise quantitative analysis of the 5-methylcytosine to cytosine ratio in a sample.

[0022] The third aspect of this application provides a kit for pretreatment of samples containing 5-methylcytosine and cytosine, comprising: magnetic beads containing sulfonic acid groups for extracting 5-methylcytosine and cytosine from the sample; an eluent (alcoholic solution) for eluting the analyte-magnetic bead complex; and an eluent (at least one of formic acid-alcohol solution and ammonia-alcohol solution) for eluting the analyte from the analyte-magnetic bead complex. This kit allows for the simple, low-cost, and efficient acquisition of analyte samples containing 5-methylcytosine and cytosine, which can then be used for highly sensitive and accurate quantitative analysis of the 5-methylcytosine to cytosine ratio using ion mobility spectrometry. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a graph showing the instrument detection results of the supernatant after the sample was adsorbed by different magnetic beads in the embodiments of this application.

[0025] Figure 2 This is a graph showing the detection results of samples being rinsed with different rinsing solutions after being adsorbed by magnetic beads in the embodiments of this application.

[0026] Figure 3 This is a graph showing the detection results of the effect of different injection solvents on the adsorption effect during sample pretreatment in the embodiments of this application.

[0027] Figure 4 This is a graph showing the detection results of the separation degree test between cytosine and 5-methylcytosine in the embodiments of this application.

[0028] Figure 5 These are the detection result diagrams corresponding to different instrument sample introduction modes in the embodiments of this application.

[0029] Figure 6 This is a graph showing the comparison and detection results of the instrument parameters optimization for the ion source voltage in the embodiments of this application.

[0030] Figure 7 This is a graph showing the comparison and detection results of instrument parameter optimization for ion gate voltage in the embodiments of this application.

[0031] Figure 8 This is a graph showing the comparison of instrument parameter optimization results for the ion gate voltage pulse width in the embodiments of this application.

[0032] Figure 9This is a graph showing the comparison of instrument parameter optimization results for the migration gas and exhaust gas flow rates in the embodiments of this application.

[0033] Figure 10 This is a graph showing the detection results of calibrator solutions with different 5-methylcytosine to cytosine ratios in the embodiments of this application.

[0034] Figure 11 This is a standard curve obtained from the detection method of the 5-methylcytosine to cytosine ratio in the embodiments of this application. Detailed Implementation

[0035] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0036] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0037] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0038] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0039] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0040] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0041] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0042] Establishing a rapid, accurate, and efficient quantitative analysis method for the 5-methylcytosine to cytosine ratio (expressed as the 5-mC / C ratio) is crucial for clinical diagnosis and disease progression monitoring. However, existing methods generally suffer from limitations such as long detection cycles, high costs, and cumbersome method switching, making it difficult to meet the needs of rapid clinical testing.

[0043] Electrospray ion mobility spectrometry (ESI-IMS) is an analytical method that combines electrospray ion sources and ion mobility spectrometry techniques for the rapid detection of trace chemical components. High-resolution electrospray ion mobility spectrometry (HPIMS) is generally used, offering advantages such as miniaturization, rapid detection, high sensitivity, simple method development, and the requirement of only trace amounts of solvent. However, there is currently no suitable detection system for the quantitative analysis of 5-mC / C.

[0044] This application aims to achieve high-throughput screening and accurate quantification of the 5-mC / C ratio in clinical practice. It develops a sample pretreatment method containing 5-methylcytosine and cytosine, and based on this pretreatment method, a detection method and related kits for the 5-mC / C ratio. This allows for accurate quantitative detection of 5-methylcytosine and cytosine via ion mobility spectrometry through direct injection without chromatographic separation, thereby improving the accuracy and throughput of the 5-mC / C ratio detection. The specific technical solution is as follows.

[0045] This application provides a sample pretreatment method, including: S01: Provide a sample containing analytes, including 5-methylcytosine and cytosine.

[0046] S02: Mix magnetic beads containing sulfonic acid groups with the sample for adsorption treatment, and then rinse with alcohol solution to obtain the analyte-magnetic bead complex.

[0047] S03: The analyte-magnetic bead complex is eluted with an eluent (including at least one of formic acid-alcohol solution and ammonia-alcohol solution) to obtain the test sample.

[0048] Magnetic beads with sulfonic acid groups on their surface are magnetic beads with a core of tiny beads made of magnetic material and a surface modified with sulfonic acid groups. The sulfonic acid group -SO3H, as an exchangeable group, is its core functional part, possessing strong cation exchange ability. Normally, before use, the magnetic beads contain negatively charged sulfonate ions -SO3H... - It can bind with hydrogen ions. When magnetic beads are mixed with samples for adsorption treatment, the 5-methylcytosine and cytosine in the sample are positively charged. These sulfonic acid groups can undergo ion exchange reactions with cations in the sample solution, so that the 5-methylcytosine and cytosine ions in the sample bind to the magnetic beads, thereby achieving the separation and purification of the analytes in the sample.

[0049] In this embodiment, magnetic beads with sulfonic acid groups on their surface are first added to the sample for adsorption extraction. 5-methylcytosine and cytosine (generally existing in cationic form) in the sample can bind to the magnetic beads via ionic bonds through cation exchange. Then, the sample is eluted with an alcohol solution to obtain the analyte-magnetic bead complex. Subsequently, at least one of formic acid-alcohol solution and ammonia-alcohol solution is used as the elution solution to remove the analyte from the magnetic beads, yielding a sample containing 5-methylcytosine and cytosine. This sample pretreatment method is not only simple and low-cost, but also efficiently yields a sample containing 5-methylcytosine and cytosine. It can then be used for highly sensitive and accurate quantification of the 5-methylcytosine to cytosine ratio using ion mobility spectrometry, thus showing great promise for the detection of the 5-methylcytosine to cytosine ratio, especially in DNA methylation detection.

[0050] In some embodiments, the sample containing the analyte provided in step S01 may contain 5-methylcytosine and cytosine, which may be free bases released after DNA degradation. For example, the bases may be released by chemical or enzymatic methods on the DNA: in embodiments, the DNA may be completely degraded using methods more conventional in the art, such as strong acid hydrolysis or specific DNA enzymes (such as deoxyribonuclease), to release the free bases.

[0051] Depending on the source of the DNA sample, the DNA can be from at least one of urine, plasma, serum, or whole blood samples. Specifically, it can be human urine or blood. The analyte originates from the bases released after DNA degradation in human urine or blood. Based on the sample pretreatment, the test sample can be obtained and subsequently used for highly sensitive detection of the 5-methylcytosine to cytosine ratio using ion mobility spectrometry, thereby achieving quantitative analysis of the 5-methylcytosine to cytosine ratio in urine or blood samples (plasma, serum, whole blood).

[0052] In some embodiments, the sample containing the analyte (5-methylcytosine and cytosine) provided in step S01 may, depending on the function of the sample, include a standard curve sample, a quality control sample, and an analytical sample arranged in parallel. The standard curve sample refers to a calibrator, which is a working solution of calibrator solutions with different 5-methylcytosine to cytosine ratio gradients prepared by dissolving 5-methylcytosine and cytosine standards in a solvent such as methanol. Further, the calibrator may also be a calibrator solution prepared by adding a matrix such as blank serum to the calibrator working solution for preparing a standard curve. This can be used to obtain a standard curve of the 5-methylcytosine to cytosine ratio, serving as a measurement benchmark for calibrating instruments, establishing standard curves, or evaluating methods. The quality control sample preparation process is similar to that of the standard curve sample, although some concentrations differ. The quality control sample is mainly used to monitor the stability and accuracy of the detection process. The analytical sample is the sample for which the 5-methylcytosine to cytosine ratio needs to be analyzed in the actual process.

[0053] Furthermore, blank samples may also be included, such as double blank samples and residue evaluation samples, which are replaced with blank matrix.

[0054] In some embodiments, the magnetic beads used in step S02 include a mixed strong cation exchange adsorbent (MCX) with a magnetic core coated on the surface of the magnetic core. The mixed strong cation exchange adsorbent includes benzenesulfonic acid groups and hydrophobic groups (e.g., phenyl or C16~C18 alkyl chains, mainly phenyl in this embodiment). This dual adsorption, through the ionic bond adsorption of the sulfonic acid groups and the reverse-phase retention by the hydrophobic interaction of the hydrophobic groups, can better extract 5-methylcytosine and cytosine.

[0055] The magnetic core consists of tiny beads made of magnetic materials (such as iron oxide), and the surface of the mixed strong cation exchange adsorbent is a porous polymer containing sulfonic acid groups and hydrophobic groups. The magnetic core and the mixed strong cation exchange adsorbent form a core / shell structure porous microsphere with a large specific surface area. Moreover, based on strong cation exchange and reverse-phase retention, it can effectively extract the analytes 5-methylcytosine and cytosine from the sample.

[0056] In some embodiments, the magnetic beads comprise a polystyrene-divinylbenzene copolymer (PS-DVB) with sulfonic acid groups bonded to the surface of the magnetic core coated with a magnetic core; that is, the surface of the magnetic beads is based on the styrene-divinylbenzene copolymer as the basic framework, and the structure contains sulfonic acid groups. Therefore, the magnetic beads have phenyl and sulfonic acid groups, which can be suitable for the exchange of 5-methylcytosine with cytosine. Moreover, the main forces are ion exchange and hydrophobic interaction, with hydrogen bonding as a secondary force, which has the characteristics of high exchange capacity, good chemical stability and fast exchange rate.

[0057] In some embodiments, the average particle size of the magnetic beads is 1-15 μm. Exemplarily, the average particle size of the magnetic beads can be any of the above values ​​or within any range of both, such as 1 μm, 2 μm, 5 μm, 6 μm, 8 μm, 10 μm, 12 μm, 14 μm, 15 μm; for example, it can be 5-10 μm, or the average particle size can be 5 μm. This application embodiment uses a magnetic bead method to pretreat samples containing 5-methylcytosine and cytosine, and the magnetic beads used have an average particle size of 1-15 μm, a large specific surface area, and their main surface functional groups can better bind the target analytes 5-methylcytosine and cytosine.

[0058] In this embodiment, the extraction of 5-methylcytosine and cytosine using magnetic beads during sample pretreatment was investigated. Specifically, cytosine was tested using three types of magnetic beads listed in Table 1 below. After extracting cytosine-containing samples with different magnetic beads, the samples were tested using the instrument system described in the subsequent embodiments. By incubating the magnetic beads with the sample (such as a solvent standard) and then taking the supernatant for testing, a lower signal response in the supernatant indicated a higher adsorption and binding rate.

[0059] The results of the on-machine test are as follows Figure 1 As shown.

[0060] Table 1

[0061] The magnetic beads in Table 1 are all from Suzhou Yingruicheng Biochemical Technology Co., Ltd. Figure 1 In the diagram, PLS-1, C18-1, and MCX-1 represent the binding of PLS, C18, and MCX in water, respectively, while MCX-2 represents the binding of MCX in a 0.1% (v / v) formic acid aqueous solution. The results show that MCX exhibits the best binding effect to cytosine under the condition of loading with a 0.1% formic acid solution, achieving an adsorption rate of approximately 69%. This suggests that ionic interactions are predominant, with reverse interactions playing a secondary role. Subsequent experiments were conducted using MCX magnetic beads.

[0062] In some embodiments, after mixing the magnetic beads with the sample, the adsorption treatment time is 1 min to 2 min. Based on the characteristics of the magnetic beads, 5-methylcytosine and cytosine in the sample can be rapidly adsorbed.

[0063] In some embodiments, before adding the magnetic beads to the sample, the magnetic beads are first activated by dispersing them in an alcohol solution, and then water is added for equilibration. The alcohol solution, as a dispersion liquid, can effectively disperse the hydrophobic magnetic beads. Specifically, the alcohol solution can be an aqueous solution of methanol, ethanol, or isopropanol, and the volume percentage of the alcohol solution can be 20-55%. For example, a 20% methanol solution, a 20% ethanol solution, or a 50% isopropanol solution. Taking isopropanol as an example, the isopropanol solution can be an aqueous solution of isopropanol, and the volume percentage of isopropanol can be 45-55%, for example, 50%. Further, the magnetic bead concentration can be 10-100 mg / mL, for example, 50 mg / mL. Under these conditions, the magnetic beads are uniformly dispersed and can efficiently adsorb 5-methylcytosine and cytosine in the sample.

[0064] After dispersing the magnetic beads in an alcohol solution, methanol can be added to activate them. Methanol activation allows the functional groups on the surface of the magnetic beads to fully expand, opening adsorption channels. Then, water is added to equilibrate the magnetic beads. Water equilibration alters the environment around the beads, reducing the risk of premature elution or ineffective adsorption of the target analyte during the adsorption process.

[0065] In some embodiments, the magnetic beads and the sample are mixed in a formic acid aqueous solution with a volume percentage of 0.01% to 1% for adsorption. Exemplarily, the volume percentage of formic acid in the formic acid aqueous solution can be any of the above values ​​or within any range of both, such as 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 0.9%, 1%. For example, it can be a 0.05% to 0.5% formic acid aqueous solution.

[0066] In this application embodiment, the diluent for the adsorption process in sample pretreatment was investigated. The results showed that 0.01%~1% formic acid water can provide a good environment for the adsorption process, with 0.1% formic acid water showing better results. This 0.1% formic acid water can be used for subsequent experiments.

[0067] In some embodiments, the rinsing solution used in step S02 is an alcohol solution. Specifically, the rinsing alcohol solution can be a methanol solution.

[0068] In this application embodiment, the elution solution in sample pretreatment was investigated. Specifically, the 5-methylcytosine and cytosine (1:1) mixture sample was purified by the above-mentioned magnetic beads MCX, and three elution modes with different volume percentages of elution solution were used: (1) 0.1% formic acid solution-5% methanol solution elution, that is, first elution with 0.1% formic acid solution and then elution with 5% methanol solution; (2) 5%~10% methanol solution elution, that is, first elution with 5% methanol solution and then elution with 10% methanol solution; (3) 0.1% formic acid solution-5%~10% methanol solution elution, that is, first elution with 0.1% formic acid solution, then elution with 5% methanol solution and finally elution with 10% methanol solution. After incubating the MCX magnetic beads with the sample (a mixture of 5-methylcytosine and cytosine in equal proportions), the extracted MCX magnetic beads were eluted with the different eluents described above, and the results were analyzed. Figure 2 As shown, 0.1%FA-5%MeOH is the first elution mode mentioned above, 5%MeOH-10%MeOH is the second elution mode mentioned above, and 0.1%FA-5%MeOH-10%MeOH is the third elution mode mentioned above. The results show that the second elution mode, i.e., elution with 5% methanol solution and 10% methanol solution in sequence, has the best effect, so this condition was adopted in subsequent experiments. Thanks to the hydrophobic difference introduced by the methyl group, 5-methylcytosine was selectively enriched, which is more conducive to subsequent analysis.

[0069] In some embodiments, to better elute the analyte-magnetic bead complex, the elution alcohol solution is an aqueous solution of an organic alcohol compound, wherein the volume percentage of the organic alcohol compound is 5% to 10%. Further, taking methanol as an example, the elution process includes: first eluting with a 5% to 7% methanol solution for 1 to 2 minutes, then eluting with a 7% to 10% methanol solution for 1 to 2 minutes. This allows for rapid elution of the analyte-magnetic bead complex from the sample.

[0070] For example, rinse with a 5% (v / v) methanol solution for 1 minute, and then rinse with a 10% (v / v) methanol solution for 1 minute.

[0071] In some embodiments, the eluent used in the elution process in step S03 is at least one of formic acid-alcohol solution and ammonia-alcohol solution.

[0072] To better elute the analyte from the magnetic beads, the volume percentage of formic acid in the formic acid-alcohol solution can be 0.00001% to 1%. For example, the volume percentage of formic acid in the formic acid-alcohol solution can be any of the following values ​​or within any range: 0.00001%, 0.0001%, 0.0005%, 0.0008%, 0.001%, 0.005%, 0.008%, 0.01%, 0.02%, 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.5%, 0.6%, 0.8%, 1%, etc. For instance, it can be a 0.0001% to 0.01% formic acid-alcohol solution, and more specifically, a 0.0005% to 0.005% formic acid-alcohol solution. The volume percentage of ammonia in the ammonia-alcohol solution can be from 0.001% to 1%. For example, the volume percentage of ammonia in the ammonia-alcohol solution can be any of the above values ​​or within any range of two of the following: 0.001%, 0.005%, 0.006%, 0.008%, 0.01%, 0.05%, 0.08%, 0.1%, 0.2%, 0.5%, 0.6%, 0.8%, 1%. For instance, it can be from 0.001% to 0.1%, and more specifically, it can be an ammonia-alcohol solution from 0.005% to 0.05%.

[0073] Specifically, the formic acid-alcohol solution includes a formic acid-methanol solution with a volume percentage of 0.00001% to 1%; the ammonia-alcohol solution includes an ammonia-methanol solution with a volume percentage of 0.001% to 1%. The ammonia solution is an aqueous solution of gaseous ammonia, typically with a mass fraction of approximately 20% to 30%.

[0074] The above-mentioned eluent can be used as an injection solvent. For example, in this embodiment, cytosine standard intermediate solution was prepared into 5 μM standard working solutions using different injection solvents, and detection was performed using the instrument system described in the subsequent embodiments: 20 μL of each cytosine standard working solution was pipetted and detected in positive ion mode. The results are as follows... Figure 3 As shown, cytosine at 1×10 -5 The response was good in formic acid in methanol (i.e., a 0.001% (v / v) formic acid in methanol solution), and also at 1×10 -4 The ammonia-methanol solution (i.e., a 0.01% (v / v) also showed a good response. Therefore, the appropriate solvent can be selected based on the specific experiment.

[0075] In some embodiments, taking an ammonia-ethanol solution as the eluent, the elution process of the analyte-magnetic bead complex includes eluting with an ammonia-methanol solution of 0.001% to 1% (v / v) for 1 to 2 minutes. This allows for rapid elution of the analyte from the analyte-magnetic bead complex, yielding a sample containing 5-methylcytosine and cytosine. This sample can be directly used for detecting the 5-methylcytosine to cytosine ratio using ion mobility spectrometry. For example, elution with a 0.01% ammonia-methanol solution for 1 minute.

[0076] Furthermore, the sample pretreatment method of this application embodiment, when combined with an automated pretreatment instrument, requires less processing time, thereby increasing the speed of sample detection and significantly reducing costs. For example, the entire sample pretreatment method can process 32 samples in only about 10 minutes.

[0077] Based on the above sample pretreatment method in the embodiments of this application, correspondingly, the embodiments of this application also provide a kit for pretreatment of samples containing 5-methylcytosine and cytosine, including: (1) magnetic beads: the surface of which contains sulfonic acid groups, used to extract the analytes 5-methylcytosine and cytosine from the sample; (2) elution solution: an alcohol solution used to clean the analyte-magnetic bead complex; (3) elution solution: at least one of formic acid-alcohol solution and ammonia-alcohol solution used to elute the analyte from the analyte-magnetic bead complex.

[0078] The kit of this application embodiment is based on the interaction of magnetic beads, elution solution (alcohol solution), and elution solution (at least one of formic acid-alcohol solution and ammonia-alcohol solution), which can obtain test samples containing 5-methylcytosine and cytosine simply, at low cost and with high efficiency. Subsequently, the 5-methylcytosine to cytosine ratio can be quantitatively analyzed with high sensitivity and accuracy based on ion mobility spectrometry.

[0079] In some embodiments, the magnetic beads in the kit comprise a hybrid strong cation exchange adsorbent with a magnetic core coated on its surface. This hybrid strong cation exchange adsorbent includes benzenesulfonic acid groups and hydrophobic groups (e.g., phenyl groups), enabling dual adsorption through the ionic bonds of the sulfonic acid groups and the reverse-phase retention by the hydrophobic interactions of the hydrophobic groups, thus better extracting 5-methylcytosine and cytosine. The magnetic core comprises tiny beads made of magnetic materials (such as iron oxide), and the hybrid strong cation exchange adsorbent on the surface is a porous polymer containing sulfonic acid groups and hydrophobic groups. The magnetic core and the hybrid strong cation exchange adsorbent form a core / shell structure of porous microspheres with a large specific surface area. Based on strong cation exchange and reverse-phase retention, it effectively extracts the analytes 5-methylcytosine and cytosine from the sample.

[0080] In some embodiments, the magnetic beads in the kit include polystyrene-divinylbenzene copolymer (PS-DVB) with sulfonic acid groups bonded to the surface of the magnetic core coated with a magnetic core; that is, the surface of the magnetic beads is based on styrene-divinylbenzene copolymer as the basic skeleton, and the structure is bonded with sulfonic acid groups, which has the characteristics of high exchange capacity, good chemical stability and fast exchange rate.

[0081] In some embodiments, the average particle size of the magnetic beads in the kit is 1–15 μm. Exemplarily, for example, it can be 5–10 μm, such as an average particle size of 5 μm. Specifically, the magnetic beads can be MCX magnetic beads with an average particle size of 5 μm, which have a large specific surface area and can better bind the target analyte.

[0082] In some embodiments, the eluent in the kit contains 5% to 10% (v / v) alcoholic organic compounds. This concentration of alcohol solution provides better rinsing results. For example, if the alcohol solution is a methanol solution, the rinsing is performed twice: first, rinse with a 5% to 7% (v / v) methanol solution for 1 to 2 minutes, and then rinse with a 7% to 10% (v / v) methanol solution for 1 to 2 minutes.

[0083] In some embodiments, the eluent in the kit is at least one of a formic acid-alcohol solution and an ammonia-alcohol solution. To better elute the analyte from the magnetic beads, the formic acid-alcohol solution may contain 0.00001% to 1% formic acid by volume, and the ammonia-alcohol solution may contain 0.001% to 1% ammonia by volume. Specifically, the formic acid-alcohol solution comprises a formic acid-methanol solution with a volume percentage of 0.00001% to 1%; the ammonia-alcohol solution comprises an ammonia-methanol solution with a volume percentage of 0.001% to 1%. This concentration of alcohol solution provides better elution. For example, elution with a 0.001% to 1% ammonia-methanol solution by volume for 1 to 2 minutes.

[0084] In some embodiments, the kit further includes an alcohol solution for dispersing and activating the magnetic beads. Specifically, the alcohol solution can be an aqueous solution of methanol, ethanol, or isopropanol, and the volume percentage of the alcohol solution can be 20-55%. For example, a 20% methanol solution, a 20% ethanol solution, or a 50% isopropanol solution. Taking isopropanol as an example, the isopropanol solution can be an aqueous solution of isopropanol, and the volume percentage of isopropanol can be 45-55%. Further, the concentration of the magnetic beads in the alcohol solution can be 10-100 mg / mL. Under these conditions, the magnetic beads are uniformly dispersed. After dispersing the hydrophobic magnetic beads in the alcohol solution, methanol can be added to activate the magnetic beads, and water can be used to equilibrate the magnetic beads.

[0085] This application also provides a method for detecting the ratio of 5-methylcytosine to cytosine (5-mC / C), including: T01: The sample containing the analyte is processed using the sample pretreatment method described in the embodiments of this application to obtain the sample to be tested.

[0086] T02: The ion mobility spectrometry method was used to detect the sample and obtain the ratio of 5-methylcytosine to cytosine.

[0087] The method for detecting the 5-methylcytosine to cytosine ratio provided in this application involves processing a sample containing 5-methylcytosine and cytosine using the sample pretreatment method provided in this application to obtain a test sample. Then, ion mobility spectrometry is used to detect the 5-methylcytosine to cytosine ratio in the test sample. This detection method allows for low-cost, efficient, and highly sensitive precise quantitative analysis of the 5-methylcytosine to cytosine ratio in a sample.

[0088] In some embodiments, the pretreatment method for the sample containing the analyte in step T01 can be found above.

[0089] In some embodiments, the ion mobility spectrometry in step T02 includes electrospray ion mobility spectrometry.

[0090] Electrospray ion mobility spectrometry (ESI-IMS) is used to detect samples containing 5-methylcytosine and cytosine, offering advantages such as rapid detection, high sensitivity, miniaturization, and low cost. The injection process eliminates the need for consumables such as chromatographic columns and mobile phases; samples are directly injected for analysis after preparation, and the detection of a single sample takes only about 1 minute. The method is simple to develop and requires only a small amount of solvent. Therefore, it can provide a rapid, low-cost, and accurate quantitative detection method for the 5-methylcytosine to cytosine ratio in clinical settings.

[0091] For example, electrospray ion mobility spectrometry (ESI-IMS) can be used to quantitatively detect the ratio of 5-methylcytosine to cytosine. 5-methylcytosine and cytosine are extracted from the sample matrix using a magnetic bead method. After sample dilution, magnetic bead extraction separation, rinsing, and elution, a directly injectable sample is prepared for analysis using ion mobility spectrometry. The ratio of 5-methylcytosine to cytosine can be accurately quantified, with a correlation coefficient of over 0.99 for the standard curve.

[0092] This application embodiment achieves accurate quantitative detection of the 5-methylcytosine to cytosine ratio by electrospray ion mobility spectrometry (ESI-IMS) through direct injection without chromatographic separation, thereby improving detection accuracy and throughput.

[0093] This application's embodiments investigated the separation degree between cytosine and 5-methylcytosine: 5 μM cytosine and 5-methylcytosine were prepared in methanol solvent and detected using the instrument system described in subsequent embodiments. The results are as follows... Figure 4 As shown, Figure 4A represents cytosine and 5-methylcytosine, which were injected separately at a concentration of 5 μM. The ion migration spectra were then combined, and the results showed that cytosine and 5-methylcytosine could be completely separated. Figure 4 Figure B in the diagram shows that the mixture of cytosine and 5-methylcytosine (both at a concentration of 5 μM) was used for analysis and was also able to separate them.

[0094] In some embodiments, ion mobility spectrometry includes electrospray ionization source ion mobility spectrometry, with detection conditions including: positive ion mode, ion source voltage 1800~2100V, migration tube voltage 8000~10000V, inlet temperature 170~240℃, migration tube temperature 170~240℃, ion gate voltage 50~60V, ion gate voltage pulse width 80~120μs, and migration gas and exhaust gas flow rates 1.0~1.5 L / min. Under the above parameters, the instrument exhibits excellent detection performance.

[0095] For example, this application embodiment tested the instrument's sample introduction mode. Specifically, 20 µL of cytosine standard working solution was aspirated and analyzed in both positive and negative ion modes. The results are as follows: Figure 5 As shown, cytosine responds better in positive ion mode, and positive ion mode was selected for all subsequent tests.

[0096] Additionally, see Figure 3 Cytosine showed good response in formic acid and methanol within a certain concentration range, and also in ammonia and methanol within a certain concentration range. Therefore, the appropriate solvent can be selected based on the specific experiment.

[0097] In some embodiments, the instrument parameters of the ion source voltage in this application are optimized: 5 μM cytosine prepared with methanol is used to optimize the ion source voltage parameters (1800V, 1900V, 2000V, 2100V, 2200V) for different ion mobility spectra. The results are as follows... Figure 6 As shown, the results indicate that cytosine exhibits the best response at an ion source voltage of 2100 V and the worst response at 2200 V. Therefore, the ion source voltage can be selected within the range of 1800–2100 V, and further sampling analysis can be performed using an ion source voltage of 2100 V.

[0098] In some embodiments, the instrument parameters of the ion gate are optimized using 5 μM cytosine prepared with methanol to optimize the parameters of ion gates (50V, 60V, 70V, 80V, 90V, 100V) with different ion mobility spectra. The results are as follows... Figure 7 As shown, cytosine responds better at an ion gate of 50-60 V, and subsequent sampling and analysis can be performed using an ion gate of 50-60 V.

[0099] In some embodiments, this application optimizes the instrument parameters for the ion gate voltage pulse width: 5 μM cytosine prepared with methanol is used to optimize the parameters for the ion gate voltage pulse width (80 μS, 100 μS, 120 μS, 150 μS) for different ion mobility spectra. The results are as follows... Figure 8 As shown, the response of cytosine increases with the increase of the ion-gate voltage pulse width, therefore, the range of 80–120 μs can be selected. Considering peak symmetry and resolution, 100 μs is currently considered, and all subsequent sampling analyses will use a parameter with an ion-gate voltage pulse width of 100 μs.

[0100] In some embodiments, the instrument parameters for the migration gas and exhaust gas flow rates are optimized using 5 μM cytosine prepared in methanol for different ion migration spectra (1.0 L / min, 1.1 L / min, 1.2 L / min, 1.3 L / min, 1.4 L / min). The results are as follows: Figure 9 As shown, cytosine can be used in the range of 1.0~1.4 L / min for both the migrating gas and the exhaust gas, with the best response at 1.2 L / min. Subsequently, parameters with a migrating gas and exhaust gas flow rate of 1.2 L / min can be selected for sampling and analysis.

[0101] Specifically, during the detection and testing of the ion mobility spectrometer, a liquid sample and solvent are introduced and ionized. Ion and solvent clusters enter the desolvation region and exit through the ion gate pulse. The ions are accelerated by an electric field of 8~10kV, collide with drifting gas (usually air or N2) along the way, and are separated through these interactions / collisions. The ions bombard the Faraday detector, which induces the charge converted into signal intensity (in volts).

[0102] In the embodiments of this application, the ratio of 5-methylcytosine to cytosine can be either a mass ratio or a molar ratio. For example, to make it easier to prepare a standard curve, the molar ratio can be used for experiments.

[0103] In some embodiments, the detection method has a lower limit of quantification (LOQ) of 1% (molar ratio) for the 5-methylcytosine to cytosine ratio. This application uses electrospray ion mobility spectrometry (ESI-IMS) to determine the 5-methylcytosine to cytosine ratio in a sample. The above-mentioned lower limit of quantification concentration is not only low-cost but also highly sensitive and accurate.

[0104] The embodiments of this application combine sample pretreatment methods and electrospray ionization source ion mobility spectrometry to improve the detection speed of the 5-methylcytosine to cytosine ratio, meet the needs of rapid clinical detection, and provide accurate quantitative results with low implementation costs, showing great application prospects.

[0105] The following description is based on specific embodiments.

[0106] Example 1: Detection of the 5-methylcytosine to cytosine ratio 1. Use of equipment and reagents.

[0107] The instruments include: High Performance Ion Mobility Spectrometer (GA2200); Data Acquisition and Data Processing Software (VISION); Analytical Balance (Mettler, accurate to 0.01 mg); Vortex Mixer (VM500PRO / VM500PRO); Solid Phase Extraction Magnetic Beads: MCX Magnetic Beads (Suzhou Yingruicheng Biochemical Technology Co., Ltd.); Fully Automated Magnetic Bead Sample Processor (Suzhou Yingruicheng Biochemical Technology Co., Ltd.).

[0108] Reagents include: 5-methylcytosine and cytosine (standards); methanol (MeOH, HPLC grade); purified water (Watsons).

[0109] Blank biological matrix: Blank human serum.

[0110] 2. Equipment conditions.

[0111] High-efficiency ion mobility spectroscopy conditions: Instrument model: Excellims GA2200; Ion source: ESI; Ionization mode: positive ion; Data acquisition time: 20 seconds; The instrument parameters are shown in Table 2.

[0112] Table 2 parameter Setting value Ionization mode Positive ion mode Ion source voltage 2100 V Migration tube voltage 9000 V Inlet temperature 180 ℃ Migration tube temperature 180 ℃ Ion gate voltage 60 V Gate voltage pulse width 100 μs Migrating airflow velocity 1.20 L / min Exhaust airflow velocity 1.20 L / min Number of spectral superpositions 10 Analysis time 20 s Spectrum acquisition time 30 ms sampling frequency <![CDATA[200000 s -1 ]]> 3. Solution preparation.

[0113] (1) Eluent: 1×10 -4 Ammonia-methanol solution (i.e., ammonia water volume percentage of 0.01%) is prepared by first mixing 25% ammonia water and methanol to form an ammonia-methanol solution with a 1% ammonia water volume percentage, and then diluting the 1% ammonia-methanol solution with methanol to a 0.01% ammonia-methanol solution.

[0114] (2) 1:5% methanol solution for rinsing: Measure 9500 μL of purified water into a glass bottle of appropriate volume, add 500 µL of methanol, mix well, and store at room temperature (25~30℃).

[0115] (3) Eluent 2: 10% methanol solution: Measure 9000 μL of purified water into a glass bottle of appropriate volume, add 1000 µL of methanol, mix well, and store at room temperature (25~30℃).

[0116] (3) Activation solution: methanol; take it and use it immediately.

[0117] (4) Preparation of matrix calibrator solutions (C1-C6): Use the stock solution to add to the matrix to prepare a series of 5-methylcytosine / cytosine ratios, with ratios of 1%, 2%, 4%, 6%, 8%, and 10% (specifically molar ratios).

[0118] 4. Standard testing and standard curve preparation Sample pretreatment: Pipette 25 μL of magnetic bead solution (MCX magnetic beads dispersed in isopropanol to prepare a 50 mg / mL magnetic bead solution) into an EP tube, add 500 μL of methanol for activation, vortex for 1 min, and magnetically discard the waste liquid; add 500 μL of H2O for equilibration, vortex for 1 min, and magnetically discard the waste liquid. Then, add 20 μL of matrix-spikened sample and 180 μL of 0.1% formic acid aqueous solution, vortex for 1 min, and magnetically discard the waste liquid; Eluent 1: Add 500 μL of eluent 1 (5% methanol solution), vortex for 1 min, and magnetically discard the waste liquid; Eluent 2: Add 500 μL of eluent 2 (10% methanol solution), vortex for 1 min, and magnetically discard the waste liquid; Elution: Add 200 μL of 1×10 -4 Eluent with ammonia-methanol solution (i.e., ammonia water volume percentage of 0.01%), vortex for 1 min, and magnetically aspirate the supernatant into a new EP tube, which is the sample to be tested.

[0119] (3) GA detection: Take 20 μL of the processed sample and put it into the instrument for detection. See Table 2 for instrument parameters.

[0120] Ion mobility spectra obtained for different 5-methylcytosine / cytosine ratios are shown in [reference]. Figure 10 Where Ratio 1% represents a 5-methylcytosine / cytosine molar ratio of 1%, Ratio 2% represents a 5-methylcytosine / cytosine molar ratio of 2%, Ratio 4% represents a 5-methylcytosine / cytosine molar ratio of 4%, Ratio 6% represents a 5-methylcytosine / cytosine molar ratio of 1%, Ratio 8% represents a 5-methylcytosine / cytosine molar ratio of 8%, and Ratio 10% represents a 5-methylcytosine / cytosine molar ratio of 10%. The linear relationship is examined as shown in Table 3 below, and the standard curve is shown in... Figure 11 As shown in the diagram. Additionally, the same steps and procedures apply to quality control samples, analytical samples, blank samples, and residue evaluation samples.

[0121] Table 3 sample 5-Methylcytosine / cytosine ratio (%) Peak area ratio (%) Accuracy (%) C1 1 2.17 96.32 C2 2 3.90 105.84 C3 4 7.07 105.56 C4 6 9.96 102.40 C5 8 12.68 99.34 C6 10 14.34 90.54 In addition, the above method can be used to further detect the actual analytical sample. Based on the ion migration spectra of different ratios of 5-methylcytosine / cytosine, the global DNA methylation level of the actual analytical sample can be calculated as [A(5mC) / (A(5mC) + A(C))]×100%; where A(5mC) represents the integral area of ​​the 5-methylcytosine peak and A(C) represents the integral area of ​​the cytosine peak.

[0122] The above embodiments illustrate that the present application combines sample pretreatment methods and electrospray ionization source high-efficiency ion mobility spectrometry to improve the detection speed of the 5-methylcytosine to cytosine ratio, meeting the needs of rapid clinical detection, and has the advantages of accurate quantitative results, high sensitivity, and low implementation cost.

[0123] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A sample pretreatment method, characterized in that, include: Provide a sample containing an analyte, said analyte including 5-methylcytosine and cytosine; The sample was mixed with magnetic beads containing sulfonic acid groups for adsorption treatment, and then washed with an alcohol solution to obtain the analyte-magnetic bead complex. The analyte-magnetic bead complex is eluted with an eluent to obtain the test sample; the eluent includes at least one of formic acid-alcohol solution and ammonia-alcohol solution.

2. The sample pretreatment method as described in claim 1, characterized in that, The magnetic beads comprise a polystyrene-divinylbenzene copolymer with a magnetic core coated on the surface of the magnetic core and bonded with sulfonic acid groups; And / or, the magnetic beads comprise a hybrid strong cation exchange adsorbent with a magnetic core coated on the surface of the magnetic core, the hybrid strong cation exchange adsorbent comprising benzenesulfonic acid groups and hydrophobic groups; And / or, the average particle size of the magnetic beads is 1~15μm.

3. The sample pretreatment method as described in claim 1, characterized in that, The alcohol solution includes a methanol solution with a volume percentage of 5% to 10%; And / or, the formic acid-alcohol solution in the eluent comprises a formic acid-methanol solution with a volume percentage of 0.00001% to 1%; And / or, the ammonia-alcohol solution in the eluent comprises an ammonia-methanol solution with a volume percentage of 0.001% to 1%.

4. The sample pretreatment method according to any one of claims 1-3, characterized in that, The rinsing treatment with alcohol solution includes: first rinsing with a methanol solution of 5%~7% by volume for 1min~2min, and then rinsing with a methanol solution of 7%~10% by volume for 1min~2min. And / or, the elution treatment of the analyte-magnetic bead complex with the elution buffer includes: eluting with an ammonia-methanol solution of 0.001%~0.1% by volume for 1 min~2 min.

5. The sample pretreatment method according to any one of claims 1-3, characterized in that, Before mixing the magnetic beads with the sample, the magnetic beads are first activated, and then water is added for equilibration. And / or, the magnetic beads are mixed with the sample in a formic acid solution with a volume percentage of 0.01% to 1% formic acid for the adsorption treatment.

6. The sample pretreatment method according to any one of claims 1-3, characterized in that, The analyte is derived from free bases released after DNA degradation; And / or, the sample containing the analyte includes a standard curve sample, a quality control sample, and an analytical sample arranged side by side.

7. A method for detecting the ratio of 5-methylcytosine to cytosine, characterized in that, include: The sample containing the analyte is processed using the sample pretreatment method according to any one of claims 1-6 to obtain the sample to be tested. The sample was analyzed by ion mobility spectrometry to obtain the ratio of 5-methylcytosine to cytosine.

8. The detection method as described in claim 7, characterized in that, The ion mobility spectrometry method includes electrospray ionization source ion mobility spectrometry, and the detection conditions include: positive ion mode, ion source voltage 1800~2100V, migration tube voltage 8000~10000V, inlet temperature 170~240℃, migration tube temperature 170~240℃, ion gate voltage 50~60V, ion gate voltage pulse width 80~120μs, and migration gas and exhaust gas flow rates 1.0~1.5 L / min; And / or, the detection method has a lower limit of quantification of 1% for the 5-methylcytosine to cytosine ratio.

9. A kit for pretreatment of samples containing 5-methylcytosine and cytosine, characterized in that, include: Magnetic beads: contain sulfonic acid groups on their surface; Rinse solution: alcohol solution; Eluent: at least one of formic acid-alcohol solution and ammonia-alcohol solution.

10. The kit according to claim 9, characterized in that, The magnetic beads comprise a polystyrene-divinylbenzene copolymer with a magnetic core coated on the surface of the magnetic core and bonded with sulfonic acid groups; And / or, the magnetic beads comprise a hybrid strong cation exchange adsorbent with a magnetic core coated on the surface of the magnetic core, the hybrid strong cation exchange adsorbent comprising benzenesulfonic acid groups and hydrophobic groups; And / or, the average particle size of the magnetic beads is 1~15μm.

11. The kit as described in claim 9, characterized in that, The alcohol solution includes a methanol solution with a volume percentage of 5% to 10%; And / or, the formic acid-alcohol solution in the eluent comprises a formic acid-methanol solution with a volume percentage of 0.00001% to 1%; And / or, the ammonia-alcohol solution in the eluent comprises an ammonia-methanol solution with a volume percentage of 0.001% to 1%.

12. The kit according to any one of claims 9-11, characterized in that, The kit also includes formic acid solution with a volume percentage of 0.01% to 1% for diluting the magnetic beads and the sample for adsorption; And / or, the kit may further include an alcohol solution for dispersing and activating the magnetic beads.

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