Method for evaluating molecular diversity of soil organic carbon
By loading conductive tape on the target plate and combining with MALDI-TOF MS technology, the problem of characterizing large molecular weight components of soil organic carbon is solved, and the accuracy evaluation of the diversity of soil organic carbon molecules is achieved, and the analysis efficiency and repetition are improved.
Patent Information
- Application Number
- CN202510291260.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-07-04
AI Technical Summary
Existing mass spectrometry technology is difficult to fully characterize the molecular diversity of soil organic carbon, especially large molecular weight and insoluble components, resulting in inaccurate analysis results and poor repeatability.
Using MALDI-TOF MS technology, soil samples were mixed with standard substances by loading conductive tape on the target plate, and mass spectrometry was performed after treatment with appropriate matrix solution, and DR and DH(P) were calculated to evaluate the molecular diversity of soil organic carbon.
It realizes simple and quick characterization of high molecular weight component information of solid soil samples, improves analysis efficiency and repetition, is suitable for high-throughput measurement, and provides a more comprehensive perspective on soil organic carbon research.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical fields of soil science and environmental science, and particularly relates to a method for evaluating the molecular diversity of soil organic carbon. Background Art
[0002] Soil organic matter constitutes the most crucial component of terrestrial ecosystems. It encompasses various plant and animal remains, microbial remains in the soil, as well as the organic carbon generated during the decomposition and synthesis of these organisms. Specifically, soil organic carbon consists of soluble components (with a molecular weight range of 1 - 5000 Da, i.e., dissolved organic carbon, which can dissolve in water or organic solvents) and insoluble components (with a molecular weight range > 5000 Da, i.e., particulate organic carbon and mineral-bound organic carbon, which cannot dissolve in water or organic solvents), and is an organic carbon with a wide molecular weight distribution and complex components. The research on the stability and molecular diversity of soil organic carbon helps soil scientists, environmental scientists, and geologists to increasingly deepen their understanding of soil organic matter.
[0003] Currently, the characterization methods of soil organic carbon are mainly divided into three categories: The first category is through imaging techniques, such as scanning electron microscopy (SEM) and 13 13C-labeled nanoprobes (Nanosims), etc., to achieve an intuitive presentation of soil organic matter; the second category is to use Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) techniques to classify the carbon functional groups and carbon structures in soil organic matter; the third category is to adopt high-resolution mass spectrometry techniques, such as Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), pyrolysis-gas chromatography-mass spectrometry (pyGC-MS), liquid chromatography-mass spectrometry (LC-MS), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), etc., to detail the characterization of the components of soil organic carbon. Among them, the third category of mass spectrometry-based techniques is also a commonly used technique for evaluating the molecular diversity of soil organic carbon.
[0004] For the third category of techniques involving FT-ICR-MS, GC-MS, LC-MS, and MALDI-based techniques, the analysis process generally includes: 1) weighing the soil sample; 2) taking the supernatant after washing the weighed soil multiple times with water, organic solvents, or reagent mixtures. During the washing process, steps such as vortexing, sonication, centrifugation, drying and reconstitution are often used to ensure that the soluble components in the soil are dissolved in the supernatant to the greatest extent; 3) taking the supernatant for data acquisition on the instrument; 4) data analysis. The characteristics of these methods are that the solid soil sample is first pretreated to obtain a liquid sample (supernatant), and then analyzed and detected. Therefore, the analysis results actually only characterize the soluble components in soil organic carbon, lacking the characterization of insoluble components, which are the main components of soil organic carbon; moreover, due to limitations such as technical methods and instrument performance, the molecular weights of the analytes are almost all within 1000 Da (<800 Da), while the molecular weight distribution of soil soluble components can be as high as 5000 Da, lacking the characterization of the molecular weight and component information of macromolecular organic carbon. Therefore, the existing methods for characterizing the components of soil organic carbon using high-resolution mass spectrometry in the third category far from meet the requirements of presenting the characteristics of soil organic carbon completely and comprehensively, which leads to inaccurate conclusions when applying the results obtained by high-resolution mass spectrometry to solve soil problems.
[0005] MALDI-TOF MS is a soft ionization technique that is good at analyzing macromolecules such as proteins, polypeptides, nucleic acids, polymers, etc. Its conventional sample pretreatment method before loading is to drop 0.5 - 1 μl of liquid sample (solution) on a stainless steel target plate, let it dry naturally, and then cover it with an equal amount of matrix. The matrix is a small molecule compound that can absorb ultraviolet light to help ionize the sample. There are many types of matrices, and different matrices have different excitation effects on different sample molecules. For example, α-cyano-4-hydroxycinnamic acid (CHCA) is generally used to analyze compounds with lower molecular weights, while sinapic acid (SA) is generally used to analyze compounds with higher molecular weights.
[0006] Some studies have shown that MALDI-TOF MS can be used to directly analyze asphalt powder samples by the "water spotting" method. The difference between the "water spotting" method and the traditional sample loading method is that the components in asphalt are insoluble in water (solubility < 10 -9) "Water" is not a solvent for asphalt but acts as an adhesive to form a film of asphalt. Therefore, the "water-droplet sampling method" enables the direct analysis of solid asphalt powder. Its technical process includes: 1) Mixing the matrix (if used) with asphalt; 2) Manually grinding and sieving the mixture to obtain a uniform powder; 3) Adding several drops of water to the powder, picking up the formed water film and transferring it to the target plate. After adding water droplets on it, use a pipette to suck the water from the bottom of the film, and after drying, perform tests on the machine. The molecular structure of asphalt contains benzene rings, which can act as the matrix by themselves, that is, ionization can be achieved without adding a matrix.
[0007] Currently, the molecular weight of asphalt samples detected by the "water-droplet sampling method" can reach 3000 - 4000 Da, and it can characterize non-soluble components, providing a technical reference for the direct analysis of non-soluble components in soil using MALDI-TOF MS. However, due to: 1) The soil contains soluble components and cannot form a water film; 2) The particle size of the soil is not standardized, and uneven particle size will cause it to be unable to be evenly distributed in the water droplets, and uneven distribution will cause deviation in the mass measurement results and inaccurate qualitative analysis; 3) It is impossible to quantify the soil particles dispersed in 1 μl of water droplets, resulting in differences in the sample loading amount between parallel samples, poor repeatability, and the results cannot be used for comparison between parallel samples; 4) Since the water acting as an adhesive is only 1 μl, it is very easy to cause cross-contamination during the sampling process; 5) MALDI-TOF MS is in a vacuum environment. When collecting data, the water acting as an adhesive has dried, and the particulate matter at the micron and nanometer levels will be free from the target plate and distributed in the vacuum tube of the instrument, causing pollution and damage to the instrument; 6) In the existing technology, even when using MALDI-TOF MS and the water-droplet sampling method, the upper limit of the analysis range of the sample molecular weight only reaches 4000 Da, which cannot meet the analysis requirements of high molecular weight soil samples.
[0008] In view of this, the present invention is specifically proposed. Summary of the Invention
[0009] The object of the present invention is to provide a method for evaluating the molecular diversity of organic carbon using MALDI-TOF MS technology. This method enables the direct characterization of the large molecular weight (up to 10 5 Da) and component information of solid soil samples, and is more simple, fast, and can be used to evaluate the molecular diversity index of soil organic carbon.
[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0011] The present invention provides a method for evaluating the molecular diversity of soil organic carbon, including the following steps:
[0012] S1. Mix the solid soil sample with the reference material, then load it onto the target plate with conductive adhesive tape and compact it.
[0013] S2. Drop the matrix solution onto the surface of the compacted sample, and dry it to obtain the sample for analysis.
[0014] S3. Use a MALDI-TOF-MS mass spectrometer to test the sample for analysis, and calculate D R and D H (P) according to the spectrum. Evaluate the molecular diversity of organic carbon in the soil sample based on the calculation results.
[0015] The step S1 is operated in one of the following ways:
[0016] 1) Press the soil sample into a tablet, load it onto the target plate with conductive adhesive tape, drop the solution containing the reference material, dry it, and compact it.
[0017] 2) Press the soil sample into a tablet, load it onto the target plate with conductive adhesive tape, sprinkle the reference material powder, and compact it.
[0018] 3) Mix the soil sample with the reference material, press it into a tablet, load it onto the target plate with conductive adhesive tape, and compact it.
[0019] 4) Spread the soil sample on the target plate with conductive adhesive tape, drop the solution containing the reference material, dry it, and compact it.
[0020] 5) Spread the soil sample on the target plate with conductive adhesive tape, sprinkle the reference material powder, and compact it.
[0021] 6) Mix the soil sample with the reference material and then spread it on the target plate with conductive adhesive tape, and compact it.
[0022] In the present invention, the soil sample is pretreated before use; the pretreatment includes: air-drying the soil sample, removing roots, sieving, and grinding; wherein the grinding is performed using a mortar or a ball mill; the diameter d of the sample after grinding is < 147 μm, and it can pass through a 100-mesh sieve.
[0023] In step S1, a sample tablet with a thickness of 1.5 - 2 mm is obtained through the pressing. The surface of the sample tablet is flat, firm, and does not loosen, and it can be picked up using tools (such as forceps).
[0024] In step S1, the selection principle of the reference material: the molecular weight distribution of the reference material should cover the molecular weight distribution of each component in the soil sample.
[0025] In a specific embodiment of the present invention, for the molecular weight range < 10 kDa, the reference substance is selected from one or more of leucine enkephalin, methionine enkephalin, bradykinin, gramicidin, neurokinin P, human renin, porcine renin, insulin A, insulin B, melittin, glucagon; for the molecular weight range > 10 kDa, the reference substance is selected from one or more of cytochrome C, bovine serum albumin.
[0026] In step S1, the mass ratio of the soil sample to the reference substance is 1:(1 - 20). Specifically, it can be adjusted according to actual needs to make the mass spectrometry peak intensity of the reference substance as consistent as possible with the peak intensities of the components of the soil sample.
[0027] In step S1, the conductive adhesive tape is a polymer fabric containing a conductive substance.
[0028] The conductive substance is selected from one or more of carbon powder, graphite powder, carbon nanotubes, and metals; wherein the metal is selected from one or more of aluminum, silver, copper, and nickel.
[0029] The polymer fabric is selected from one or more of polyester fiber, polyester fiber, and nylon.
[0030] The thickness of the conductive adhesive tape is 30 - 45 μm, and the resistivity < 5 OHMS / mm 2 。
[0031] In step S2, the matrix is selected according to the following conditions:
[0032] When the molecular weight of the soil sample < 5 kDa, the matrix is α-cyano-4-hydroxycinnamic acid; the concentration of α-cyano-4-hydroxycinnamic acid in the solution is 5 - 20 mg / ml.
[0033] When the molecular weight of the soil sample is between 5 kDa and 100 kDa, the matrix is sinapic acid. The concentration of sinapic acid in the solution is 15 - 35 mg / ml.
[0034] In step S2, the solvent used for the matrix solution is a mixed solution of acetonitrile, water, and trifluoroacetic acid; wherein, the volume content of trifluoroacetic acid is 0.75% - 2.5%, and the volume content of acetonitrile is 50% - 70%.
[0035] In step S3, the conditions for the test mass spectrometry test are:
[0036] Ionization mode: Linear(positive mode);
[0037] Scanning range: 0 - 100 kDa;
[0038] Spots: 1 spot; Accumulate: 10 shot(s);
[0039] Profiles: 500 profiles;
[0040] Pulsed extract: 3000 (0 - 5 kDa) / 60000 (5 - 100 kDa);
[0041] Blanking: 50 (0 - 5 kDa) / 6000 (5 - 100 kDa);
[0042] Laser intensity: 80 - 100 ((0 - 5 kDa) / 120 - 140 (5 - 100 kDa).
[0043] In step S3, the calculation is performed according to the following operations: Filter out valid m / z peaks from the peak list corresponding to the original spectrum according to the signal-to-noise ratio S / N > 3 and the resolution Resolution > 300, and calculate to obtain D R and D H (P).
[0044] In the present invention, according to D R and D H (P) to evaluate the diversity of soil molecules, specifically: the more D R or / and D H (P), the greater the diversity of the organic carbon molecular composition, the less likely it is to be decomposed, and the more stable the organic carbon is.
[0045] In the present invention, the quantification of the molecular diversity includes: the number of organic carbon molecular compositions (Richness) and the types of organic carbon molecular compositions (Diversity).
[0046] Calculation formula I for the number of organic carbon molecular compositions:
[0047] D R = S,
[0048] where S represents the number of valid peaks (m / z) in the mass spectrometry data;
[0049] Calculation formula II for the types of organic carbon molecular compositions:
[0050]
[0051] where P i represents the relative intensity of the valid peak, S represents the number of valid peaks, and i represents the corresponding m / z on the peak list.
[0052] In step S3, according to the calculation results, evaluate the molecular diversity of organic carbon in the soil.
[0053] The evaluation method of the molecular diversity is as follows: Based on the quantified D R and D H (P) results, the more D R or / and D H (P), the greater the diversity of the organic carbon molecular composition, the less likely it is to be decomposed, and the more stable the organic carbon is.
[0054] The present invention also provides a test kit for the molecular diversity of soil organic carbon, which comprises the following components: a target plate loaded with conductive adhesive tape, an operation manual recording the above method, a soil standard sample and a reference substance.
[0055] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:
[0056] By loading carbon cloth on the target plate, the present invention solves the difficulty of the homogeneity of solid substances, simplifies the sample preparation process, effectively avoids the destruction of samples during the analysis process, improves the analysis efficiency and speed, and is particularly suitable for high-throughput determination requirements; by further screening suitable matrices and optimizing the analysis operation steps, the present invention finally realizes the direct characterization and analysis of organic carbon in solid soil samples by using MALDI-TOF-MS technology in a wide range of 0 to 10 5 Da, overcomes the limitation that the traditional method can only measure small molecular weight organic carbon in soil, and provides a more comprehensive perspective for the research of soil organic carbon.
[0057] In summary, the qualitative and quantitative analysis method of the molecular diversity of soil organic carbon based on mass spectrometry technology provided by the present invention provides new tools and methods for soil science research, and helps to deeply understand the composition and function of soil organic carbon. Brief Description of the Drawings
[0058] Figure 1 It is a flowchart of the preparation of the sample for machine analysis in the method of the present invention.
[0059] Figure 2 It is a schematic structural diagram of the MALDI-TOF MS target plate loaded with peelable carbon cloth in the present invention.
[0060] Figure 3 It is an example of the spectrum of MALDI-TOF mass spectrometry analysis of soil solid particles loaded on conductive adhesive tape (mass range 10 - 100 kDa). Detailed Embodiments
[0061] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.
[0062] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.
[0063] The reagents, materials, instruments, etc. used in the following examples can be obtained from commercial sources without special instructions.
[0064] Example, Molecular Diversity Analysis of Soil Organic Carbon
[0065] 1. Sample Collection:
[0066] The collection site is the Eerguna Meadow Steppe in Inner Mongolia. Extract the mineral-bound organic matter (3 replicates) from the surface soil (0 - 20 cm) of the grassland, air-dry it, pick out the roots, and pass it through a 100-mesh sieve after grinding.
[0067] 2. Preparation of Standard Substance Solution:
[0068] Prepare an aqueous solution of cytochrome C (from horse heart) with a final concentration of 5 pmol / μL.
[0069] 3. Preparation of Matrix Solution:
[0070] Weigh 30 mg of sinapic acid (SA) powder, add 700 μL of acetonitrile and 300 μL of 2.5% trifluoroacetic acid (TFA) solution (final volume 1 mL), vortex for about 1 minute to fully dissolve the matrix.
[0071] 4. Target Plate Preparation
[0072] (1) Direct Sampling of Soil Solid: Weigh a quantitative amount of soil sample powder (0.2 - 0.5 mg), evenly spread it on the spotting holes of the target plate with a conductive carbon cloth, and compact it. Then spot 1 μL of the matrix solution on it and let it dry naturally.
[0073] (2) Sampling by Droplet Method: Spot 1 μL of pure water on the target plate holes (without conductive carbon cloth), then distribute a quantitative amount of soil sample powder as evenly as possible on the surface of the water droplet, and let it dry naturally. Then spot 1 μL of the matrix solution on it and let it dry naturally.
[0074] (3) Sampling of Standard Substance: Drop 1 μL of the standard substance solution on the spotting holes of the target plate with a conductive carbon cloth, and let it dry naturally. Then spot 1 μL of the matrix solution on it and let it dry naturally.
[0075] (4) Blank Matrix: Drop 1 μL of the matrix solution on the spotting holes of the target plate with a conductive carbon cloth, and let it dry naturally.
[0076] 5. Use a MALDI-TOF-MS mass spectrometer to perform mass spectrometry tests on the samples to be analyzed, and calculate the molecular diversity of soil organic carbon;
[0077] Data Collection:
[0078] The conditions for the mass spectrometry test are:
[0079] Ionization instrument parameter settings: Acquisition mode: Linear (positive mode);
[0080] It is the positive ion linear mode, and the scanning range of the mass spectrum: 0 to 10 - 100 kDa;
[0081] Spots: 1 spot, Accumulate: 10 shot(s);
[0082] Profiles: The acquisition frequency is 200 Hz, and each mass spectrum consists of 500 profiles;
[0083] Pulsed extract: 3000 (0 - 5 kDa) / 60000 (5 - 10 kDa);
[0084] Blanking: 50 (0 - 5 kDa) / 6000 (5 - 10 kDa);
[0085] Each mass spectrum is composed of profiles, and each profile contains 10 shots.
[0086] Laser intensity: 80 - 100 ((0 - 5 kDa) / The energy can be adjusted according to the actual state of the instrument, such as setting the laser energy to 120 - 140% of the reference value (5 - 10 kDa).
[0087] The original mass spectrometry data is screened using molecular weight and relative peak intensity; the screening conditions are: signal-to-noise ratio (S / N) > 3, resolution > 300.
[0088] The calculation formula is:
[0089] Calculation formula I for the number of organic carbon molecular components: DR = S;
[0090] In the formula, S represents the effective peaks in the mass spectrometry data (Data acquisition steps: Insert the prepared target plate into the bench-top MALDI-TOF mass spectrometer. After the vacuum is ready, calibrate the position of the target plate, and then set the parameters according to the above requirements. First, acquire the mass spectrum of the standard substance, and use this mass spectrum to calibrate the mass axis of the instrument so that the mass deviation is within ±1 Da. At the same time, observe whether the intensity of the m / z peaks of the standard substance meets the requirements of daily quality control, evaluate the instrument state and appropriately adjust the laser energy value. Second, acquire the mass spectrum of the blank matrix to examine whether there is contamination. Third, acquire the mass spectrum of the sample prepared by the water droplet loading method. Fourth, acquire the mass spectrum of the sample prepared by the solid direct loading method.
[0091] 6. Post-processing method of the mass spectrum
[0092] The post - processing parameters are set as follows: The Smoothing option is set to Gaussian - 1 filter width; the Peak width option is set to 1; the Peak delimiter method is set to Threshold Apex - 0.0001 mV. Under such parameter processing, the MALDI - TOF mass spectrum as shown in Figure 3 can be obtained. The peak list can be extracted from the obtained MALDI - TOF mass spectrum, as shown in Table 1 below.
[0093] Table 1 Schematic diagram of the peak list corresponding to the MALDI - TOF mass spectrum
[0094] Mass (Da) Intensity (mV) Intensity (%) Area (mV) Area (%) Resolution S / N …… …… …… …… …… …… …… 10091.56 2.29 0 34.33 0 6759 50 …… …… …… …… …… …… …… 21652.4 0.18 0 0.29 0 43386 46 26803.3 0.12 0 0.24 0 42110 96 30602 0.18 0 0.29 0 51579 46 31969.9 0.18 0 0.29 0 52720 146 33726.2 0.12 0 0.32 0 22915 31 …… …… …… …… …… …… …… 86222.26 0.85 0 2.73 0 39804 152 90383.6 0.71 0 1.86 0 53665 45 …… …… …… …… …… …… ……
[0095] 7. Data analysis
[0096] (1) Screening of effective mass spectrometry peaks: In the peak list, examine the signal - to - noise ratio (S / N) and resolution (Resolution) corresponding to each m / z peak. When both S / N > 3 and Resolution > 300 are satisfied, the m / z peak is identified as an effective peak and can participate in the calculation.
[0097] (2) Influence of two sample introduction methods on the number and repeatability (RSD%) of m / z peaks: For the same soil powder sample, the conductive tape direct sample introduction method and the traditional water - drop sample introduction method are respectively used for investigation. It can be clearly observed that the conductive tape method significantly improves the homogeneity of sample distribution. Further, as can be seen from Table 2, the number of effective peaks (1800) obtained by using the conductive tape direct sample introduction method is significantly higher than that of the water - drop sample introduction method (27), and the repeatability between parallel samples is also significantly improved. This shows that this method has successfully realized the analysis of soil molecular richness (i.e., the number of effective peaks).
[0098] Table 2 Number and RSD% of m / z peaks (n = 3)
[0099] Droplet loading method Conductive tape method <![CDATA[m / z peak ＊ Quantity (n = 3)]]> 27±6 1800±32 RSD% (n = 3) 70％ 50％
[0100] ＊ S / N > 3 and Resolution > 300
[0101] (3) Calculation of soil molecular diversity: Based on the number and intensity of mass spectrometry peaks, calculate the molecular richness (Richness, i.e., the number of detected peaks) and diversity (D H index, ). The results are shown in Table 3.
[0102] Table 3 Calculation results of soil molecular diversity (n = 3)
[0103]
[0104] These results show that by using MALDI-TOF mass spectrometry and adopting a target plate preparation method based on conductive adhesive tape, direct analysis of soil solid particles can be achieved, and the molecular abundance in the high molecular weight distribution range (such as 10 - 100 kDa) of soil components can be effectively obtained. This method is currently the only method in the field of soil science that can be used to evaluate the molecular diversity of soil components in the high molecular weight range.
[0105] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.
Claims
1. A method for evaluating the molecular diversity of soil organic carbon, comprising the following steps: S1. Mix the solid soil sample with a standard substance, then place it on a target plate with conductive adhesive tape and compact it; S2. Drop a matrix solution onto the surface of the compacted sample and dry it to obtain a sample for analysis; S3. Use a MALDI-TOF-MS mass spectrometer to test the sample to be analyzed, and calculate D based on the spectrum R and D H (P), and evaluate the molecular diversity of organic carbon in the soil sample according to the calculation results.
2. The method according to claim 1, wherein The step S1 is operated in one of the following ways: 1) Press the soil sample into a tablet, place it on a target plate with conductive adhesive tape, drop a solution containing the standard substance, dry it, and then compact it; 2) Press the soil sample into a tablet, place it on a target plate with conductive adhesive tape, sprinkle the standard substance powder, and then compact it; 3) Mix the soil sample with the standard substance, press it into a tablet, place it on a target plate with conductive adhesive tape, and then compact it; 4) Spread the soil sample on a target plate with conductive adhesive tape, drop a solution containing the standard substance, dry it, and then compact it; 5) Spread the soil sample on a target plate with conductive adhesive tape, sprinkle the standard substance powder, and then compact it; 6) Mix the soil sample with the standard substance and then spread it on a target plate with conductive adhesive tape and compact it.
3. The method according to claim 1 or 2, characterized in that, In step S1, the selection principle of the standard substance is that the molecular weight distribution of the standard substance should cover the molecular weight distribution of each component in the soil sample.
4. The method according to any one of claims 1 to 3, characterized in that, In step S1, the mass ratio of the soil sample to the standard substance is 1:(1 - 20).
5. The method according to any one of claims 1-4, characterized in that, In step S1, the conductive adhesive tape is a polymer fabric containing a conductive substance; The conductive substance is selected from one or more of carbon powder, graphite powder, carbon nanotubes, and metals; The polymer fabric is selected from one or more of polyester fiber, polyester fiber, and nylon; The thickness of the conductive adhesive tape is 30 - 45μm, and the resistivity < 5 OHMS / mm 2 .
6. The method according to any one of claims 1-5, characterized in that, In step S2, the matrix is selected according to the following conditions: When the molecular weight of the soil sample < 5 kDa, the matrix is α-cyano-4-hydroxycinnamic acid, and the concentration of α-cyano-4-hydroxycinnamic acid in the solution is 5 - 20 mg / ml; When the molecular weight of the soil sample is between 5 kDa and 100 kDa, the matrix is sinapic acid, and the concentration of sinapic acid in the solution is 15 - 35 mg / ml.
7. The method according to any one of claims 1-6, characterized in that, In step S3, the conditions for the mass spectrometry test are as follows: Ionization mode: Linear; Scanning range: 0 - 100 kDa; Spots: 1 spot Accumulate: 10 shot; Profiles: 500 profiles; Pulsed extract: If the molecular weight is between 0 - 5 kDa: 3000; if the molecular weight is between 5 - 100 kDa: 60000; Blanking: If the molecular weight is between 0 - 5 kDa: 50; if the molecular weight is between 5 - 100 kDa: 6000; Laser intensity: If the molecular weight is between 0 - 5 kDa: 80 - 100; if the molecular weight is between 5 - 100 kDa: 120 - 160.
8. The method according to any one of claims 1 to 7, characterized in that In step S3, the calculation is performed according to the following operations: screening out valid m / z peaks from the peak list corresponding to the original spectrum according to the signal-to-noise ratio S / N > 3 and the resolution Resolution > 300, and calculating to obtain D R and D H (P).
9. The method according to any one of claims 1-8, characterized in that In step S3, the quantification of the molecular diversity includes the number and types of organic carbon molecular compositions; The calculation formula I for the number of organic carbon molecular compositions: D R = S, In the formula, S represents the number of valid peaks in the mass spectrometry peak list data; The calculation formula II for the types of organic carbon molecular compositions: Wherein, P i represents the relative intensity of the effective peak, S represents the number of effective peaks, and i represents the corresponding m / z on the peak list.
10. A test kit for the molecular diversity of soil organic carbon, comprising the following components: a target plate loaded with conductive adhesive tape, an operation manual recording the method according to any one of claims 1-9, a soil standard sample, and a reference substance.