Derivatization reagent for detecting plant hormones in plants and application of derivatization reagent
By using derivatized reagents prepared by DMPI and HATU as matrix, the problems of limited matrix screening and high limit of plant hormone detection in the prior art are solved, and efficient detection of a variety of plant hormones and good repeat stability performance are achieved.
Patent Information
- Application Number
- CN202411863717.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-04-29
AI Technical Summary
In the prior art, when detecting plant hormones, matrix screening is limited, and the detection of most plant hormones is limited, so it is impossible to efficiently detect multiple plant hormones.
The derivatization reagent prepared by DMPI and HATU dissolved in aqueous acetonitrile and filtered through ultrasonic and hydrophobic filter membranes was sprayed on plant tissue samples as a matrix for mass spectrometry detection.
It realizes efficient detection of a variety of plant hormones, improves detection types and accuracy, and has good repeatability and stability.
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Figure CN119901551A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant hormone determination, and particularly relates to a derivatization reagent for detecting plant hormones in plants and an application thereof. Background Art
[0002] Plant hormones are a type of organic compound that is present in very low amounts in plants but plays an important role in regulating plant growth and development and other life processes. Detecting the spatial distribution characteristics of plant hormones is of great significance for studying plant growth and development. Currently, the matrix-assisted laser desorption ionization (MALDI) method is commonly used to detect plant hormones. MALDI is a soft ionization method, and its "matrix" is determined by the type of molecule to be detected. Usually, an excess of the matrix is added to the sample to be analyzed and laser irradiation is used to vaporize the analyte molecules to produce positively and negatively charged ions. It has the advantages of being fast, accurate, and having high resolution, and is currently one of the most commonly used technical means in the field of mass spectrometry imaging.
[0003] Matrix screening is one of the key steps in evaluating the quality of MALDI imaging results. The main reason is that in the matrix-assisted laser desorption ionization analysis process, in the low molecular weight region, the matrix will not only interfere with the desorption of the sample, but the matrix itself will also be ionized, thereby generating a large number of molecular ion peaks, fragment peaks and matrix cluster peaks, etc. These peaks often rob the sample peak of laser energy, or overlap with the sample peak, making the analysis of the analyte complicated. Therefore, screening out a matrix suitable for the detection of metabolites of interest often affects the final detection of the sample. The defects of the prior art are mainly that only a small number of plant hormones, 2-8, can be screened through the conventionally used matrix, and the detection limit of most plant hormones is much higher than the actual content of the sample. The problems that can be solved by the conventionally used matrix are often limited.
[0004] In addition, there are many types of plant hormones. Currently, there are six known types of hormones produced in plants, namely auxin, gibberellin, cytokinin, abscisic acid, ethylene and brassinosteroids. There are large differences in the chemical properties between them. For example, the polarity, chemical structure and properties of different hormones, the distribution area and content in different plant samples are quite different. Derivatization is an effective technical means, but the existing derivatization has some problems such as complex operation and poor solid derivatization effect. For example, the 3-NPH derivatization reagent is first sprayed on the surface of the glass slide. After the derivatization is completed, 5-10 layers of DHB matrix-assisted ionization are sprayed on the surface of the glass slide. The operation is not only complicated, but also has poor detection effect for small molecule compounds such as plant hormones. Katsuhiro Shiono et al. used the matrix CHCA to detect two plant hormones, trans-zeatin and abscisic acid, in rice roots. Katsuhiro Shiono used a new nanomaterial matrix to detect 8 plant hormones. At present, there is a lack of a feasible technology that can efficiently detect multiple plant hormones. Summary of the invention
[0005] In view of this, the present invention provides a derivatization reagent for detecting plant hormones in plants and application thereof.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions: A derivatization reagent for detecting plant hormones in plants is prepared by dissolving DMPI and HATU in an acetonitrile aqueous solution and filtering through ultrasound and a hydrophobic filter membrane.
[0007] Furthermore, the molar ratio of DMPI to HATU is 1:(0.6-0.8).
[0008] Furthermore, the acetonitrile concentration in the acetonitrile aqueous solution is 60-70%; the ultrasonic conditions are: 300-500W, time 4-6min; and the pore size of the hydrophobic filter membrane is 0.22μm.
[0009] A method for improving the detection of plant hormones in plants using the above-mentioned derivatization reagent comprises the following steps: S1. Take a plant tissue sample and lay it flat in an embedding box containing an embedding agent, freeze it, take the frozen and stabilized embedding block for sectioning, and place the section on an ITO glass slide; S2, adding the derivatization reagent into a matrix sprayer, and spraying it on the ITO glass slide containing the slice to obtain a sample to be tested; S3. Place the sample to be tested in a mass spectrometer for detection to obtain the plant hormone information in the plant tissue sample.
[0010] Furthermore, in step S1, the embedding agent is a carboxymethyl cellulose solution with a mass volume ratio of 2%, the freezing temperature is -80°C, and the slice thickness is 8-50 μm.
[0011] Furthermore, in step S2, 1.5-2.5 mL of the derivatization reagent is sprayed on each ITO glass slide.
[0012] In some specific embodiments, preferably, the matrix spray instrument parameters in step S2 are set as follows: temperature is 50° C., number of cycles is 25, pressure is 5 psi, and flow rate is 0.2 mL / min.
[0013] In some specific embodiments, preferably, the mass spectrometer parameters in step S3 are set as follows: m / z acquisition range 100-1000, laser energy 50%, and acquisition times 40 times.
[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) The derivatization reagent provided by the present invention can replace the matrix itself, so there is no need for additional matrix spraying to assist in the ionization of metabolites. The key role is that the reaction conditions between the derivatization reagent and the carboxylic acid group are mild, and the degree of ionization of the group after the derivatization reaction is high, which is easily detected by mass spectrometry as DMPI-R with a positive charge. + The derivatization reaction pathway is as follows: .
[0015] (2) Most of the current derivatization schemes require a high temperature, high pressure or liquid environment to react. In order to realize the characteristic of visualizing the spatial distribution of tissues, mass spectrometry imaging requires that tissues be sliced and transferred to ITO conductive glass slides during the sample pre-treatment stage, which cannot create a high temperature, high pressure or liquid environment for the slices. The derivatization reagent provided by the present invention can be used as an excellent matrix and has mild reaction conditions (50 degrees can be sprayed on the surface of the glass slide to react).
[0016] (3) A total of 39 plant hormones were developed using the derivatization reagents provided by the present invention, of which 11 substances could be confirmed using the secondary spectra of the standard substances; they had good repeatability and stability. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is the result spectrum of detecting the mixed standard of indoleacetic acid and abscisic acid in Example 1 of the present invention.
[0018] Figure 2 This is the result spectrum of detecting the mixed standard of indoleacetic acid and abscisic acid in Comparative Example 1 of the present invention.
[0019] Figure 3 , 4This is a graph showing the results of hormone testing in garlic. DETAILED DESCRIPTION
[0020] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.
[0021] Sources and physical and chemical parameters of key test materials: HATU (chemical name: 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluorophosphate; CAS number: 148893-10-1; Chinese name: 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluorophosphate) was purchased from Aladdin Reagent (Shanghai) Co., Ltd.
[0022] DMPI (chemical name: N,N-dimethylpiperazine iodide; CAS number: 106-58-1; Chinese name: N,N'-dimethylpiperazine) was purchased from Aladdin Reagent (Shanghai) Co., Ltd.
[0023] NEDC (chemical name: N-(1-Naphthyl) Ethylenediamine Dihydrochloride; CAS number: 1465-25-4; Chinese name: n-(1-naphthyl)-ethylenediamine dihydrochloride) was purchased from Aladdin Reagent (Shanghai) Co., Ltd.
[0024] The matrix sprayer is an M5-Sprayer fully automatic matrix sprayer.
[0025] The present invention utilizes a derivatization reagent to detect plant hormones, which is achieved by the following steps: S1. Dissolve DMPI and HATU in a 60-70% acetonitrile aqueous solution at a molar ratio of 1: (0.6-0.8), sonicate for 4-6 minutes at 300-500W ultrasonic conditions, and then filter through a hydrophobic filter membrane with a pore size of 0.22 μm to prepare a derivatization reagent.
[0026] S2. Take a plant tissue sample, spread it flat in an embedding box containing a 2% carboxymethyl cellulose solution in a mass-volume ratio, and freeze it at -80°C for 30 to 50 minutes. Take the frozen and stabilized embedded block for sectioning with a thickness of 8 to 50 μm, and place the section on an ITO glass slide, i.e., a plant tissue sample slide.
[0027] S3, adding the derivatization reagent in step S1 to the matrix sprayer, and spraying it on the plant tissue sample slide obtained in step S2, so as to obtain the sample slide to be tested. Wherein, each plant tissue sample slide is sprayed with 1.5-2.5 mL of the derivatization reagent; the matrix sprayer parameters are set as follows: temperature is 50°C, number of cycles is 25, pressure is 5 psi, and flow rate is 0.2 mL / min.
[0028] S4, placing the sample glass slide obtained in step S3 in a mass spectrometer for detection, so as to obtain the plant hormone information in the plant tissue sample. The mass spectrometer parameter settings are: m / z acquisition range 100-1000, laser energy 50%, and acquisition times 40 times.
[0029] Example 1 In this example, a derivatization reagent prepared by DMPI and HATU was used to test a mixed standard of indoleacetic acid and abscisic acid to verify its detection effect. The specific steps are as follows: S1. Dissolve DMPI and HATU in 60% acetonitrile aqueous solution at a molar ratio of 1:0.7, sonicate for 5 min at 400 W ultrasonic conditions, and then filter through a hydrophobic filter membrane with a pore size of 0.22 μm to prepare a derivatization reagent.
[0030] S2. Use the drip-drying deposition method to drop 1 μL of mixed standard (indoleacetic acid and abscisic acid concentrations are both 1 ppm) and 1 μL of DMPI onto the bruker target plate; adjust the mass spectrometer parameter settings for on-machine detection: m / z acquisition range 100~1000, laser energy 50%, acquisition times 40 times. Specific test results are shown in Figure 1 (Among them, a and c are the primary information spectra of the blank control groups of indoleacetic acid and abscisic acid, respectively; b and d are the secondary information spectra of indoleacetic acid and abscisic acid after adding derivatization reagents, respectively).
[0031] Depend on Figure 1 It can be seen that the derivatization reagent provided in the present application has a good detection effect on the mixed standard of indoleacetic acid and abscisic acid, and can still be detected even at a low concentration (1 ppm).
[0032] Comparative Example 1 This comparative example uses conventional 3-NPH and NEDC-configured derivatization reagents to test the mixed standard of indoleacetic acid and abscisic acid to verify its detection effect. The specific steps are as follows: S1. Dissolve 3-NPH and NEDC in 80% acetonitrile aqueous solution at a molar ratio of 1:0.7 to prepare a derivatization reagent.
[0033] S2, the steps are the same as S2 in Example 1, except that the concentrations of indoleacetic acid and abscisic acid in the mixed standard are both 50 ppm. Figure 2 (Among them, a is the information spectrum after adding the derivatization reagent; b is the information spectrum of the blank control group of indoleacetic acid and abscisic acid).
[0034] Depend on Figure 2 It can be seen that the conventional 3-NPH and NEDC-configured derivatization reagents cannot detect the mixed standard of indoleacetic acid and abscisic acid at all. In addition, this comparative example also tested the mixed standard with indoleacetic acid and abscisic acid concentrations of 1ppm, and found that they could not be detected. It is believed that the concentration of the standard may be too low, so the concentrations of indoleacetic acid and abscisic acid in the mixed standard were increased to 50ppm, but they still could not be detected. The reasons for this result may be: the derivatization reaction may require a liquid environment; the matrix NEDC added after derivatization may not be the optimal matrix.
[0035] Furthermore, during the experiment, the matrix DHB and CHCA were used for detection, and it was found that indoleacetic acid and abscisic acid could not be detected. The reason for this result may be that both matrices contain carboxylic acid groups, which will react with the derivatization reagents and thus inhibit the detection of plant hormones.
[0036] Example 2 This example uses a derivatization reagent configured with DMPI and HATU to develop detectable plant hormone types, and its operation steps are basically the same as those in Example 1, except that there are more types of standard species in the mixed standard sample in step S2. The specific plant hormone development results are shown in Table 1 below.
[0037] Table 1 Details of plant hormones developed from derivatization reagents
[0038] It can be seen from Table 1 above that the derivatization reagents provided in this application have developed a total of 39 plant hormones, including 11 gibberellins, 6 jasmonic acids, 15 auxins, 5 salicylic acids, and 2 abscisic acids.
[0039] Example 3 In this example, a derivatization reagent configured with DMPI and HATU was used to detect three plant samples, namely, rice roots, tobacco stems, and soybean roots, to verify the detection effect. The specific steps are as follows: S1. Steps are the same as S1 in Example 1.
[0040] S2. Take three kinds of plant tissue samples, namely rice root, tobacco stem and soybean root, respectively, spread them flat in an embedding box containing a 2% carboxymethyl cellulose solution in a mass-volume ratio, freeze them at -80°C for 40 min, take the frozen and stabilized embedded blocks for sectioning, with a section thickness of 10 μm, and store them on an ITO glass slide, i.e., a plant tissue sample slide.
[0041] S3, adding the derivatization reagent in step S1 to the matrix sprayer, and spraying it on the plant tissue sample slide obtained in step S2, so as to obtain the sample slide to be tested. Wherein, each plant tissue sample slide is sprayed with 2 mL of the derivatization reagent; the parameters of the matrix sprayer are set as follows: temperature is 50°C, number of cycles is 25, pressure is 5 psi, and flow rate is 0.2 mL / min.
[0042] S4, placing the sample glass slide obtained in step S3 in a mass spectrometer for detection, so as to obtain the plant hormone information in the plant tissue sample. The mass spectrometer parameter settings are: m / z acquisition range 100-1000, laser energy 50%, and acquisition times 40 times.
[0043] The specific test results of the types of plant hormones in the three plant samples of rice roots, tobacco stems, and soybean roots are shown in Table 2.
[0044] Table 2 Specific detection results of plant hormone types in three plant samples
[0045] It can be seen from the above table that the derivatization reagent provided in the present application was used to detect different types of plant samples, and more than 20 types of hormones were detected, with a high detection rate of plant hormones.
[0046] Furthermore, in order to understand the detection stability of the derivatization reagent provided in the present application, three repeatable experiments were set up for garlic for detection. The garlic samples were: garlic 0831, garlic 0905, and garlic 0911. The specific number of garlic indicates the time and date, and the detection effect of the garlic slice samples after being placed for 2 weeks was further verified. The specific test results are shown in Table 3, and the detection spectrum is shown in Figure 3 , 4 .
[0047] Table 3 Hormone detection results in three different varieties of garlic
[0048] It can be seen from the above table that the plant hormone detection results are the same in multiple tests on the same plant, and the results of re-testing the samples of the same plant after two weeks are also the same, which shows that the derivatization reagent provided by the present application has good repetitive stability.
[0049] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.
[0050] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A derivatization reagent for detecting plant hormones in plants, characterized in that: The derivatization reagent is prepared by dissolving DMPI and HATU in an acetonitrile aqueous solution and filtering through ultrasound and a hydrophobic filter membrane.
2. The derivatization reagent according to claim 1, characterized in that The molar ratio of DMPI to HATU is 1:(0.6-0.8).
3. The derivatization reagent according to claim 2, characterized in that The acetonitrile concentration in the acetonitrile aqueous solution is 60%-70%; the ultrasonic conditions are: 300-500W, time 4-6min; the pore size of the hydrophobic filter membrane is 0.22μm.
4. A method for improving the detection of plant hormones in plants using the derivatization reagent of claim 1, characterized in that: The following steps are involved: S1. Take a plant tissue sample and lay it flat in an embedding box containing an embedding agent, freeze it, take the frozen and stabilized embedding block for sectioning, and place the section on an ITO glass slide; S2, adding the derivatization reagent into a matrix sprayer, and spraying it on the ITO glass slide containing the slice to obtain a sample to be tested; S3. Place the sample to be tested in a mass spectrometer for detection to obtain the plant hormone information in the plant tissue sample.
5. The method according to claim 4, characterized in that In step S1, the embedding medium is a carboxymethyl cellulose solution with a mass volume ratio of 2%, the freezing temperature is -80°C, and the slice thickness is 8-50 μm.
6. The method according to claim 4, characterized in that In step S2, 1.5-2.5 mL of the derivatization reagent is sprayed on each ITO glass slide.
7. The method according to claim 4, characterized in that The parameters of the matrix sprayer in step S2 were as follows: temperature of 50° C., number of cycles of 25, pressure of 5 psi, and flow rate of 0.2 mL / min.
8. The method according to claim 4, characterized in that In step S3, the mass spectrometer parameters are set as follows: m / z acquisition range 100-1000, laser energy 50%, and acquisition times 40 times.