CN111912894A - Method for measuring plant rhizosphere secretion based on thin film solid phase microextraction-FT-ICR MS combination - Google Patents
Method for measuring plant rhizosphere secretion based on thin film solid phase microextraction-FT-ICR MS combination Download PDFInfo
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Abstract
A method for measuring plant rhizosphere secretion based on thin film solid phase microextraction-FT-ICR MS combined use belongs to the fields of analytical chemistry and environmental engineering. The method comprises the following steps: the method comprises the following steps: (1) collecting plant rhizosphere secretion solution; (2) extracting plant rhizosphere secretion solution by a TFME method (3) and analyzing the components of the plant rhizosphere secretion by FT-ICR MS. The invention does not need to prepare a large amount of sample solution, has simple operation process, can effectively improve the detection sensitivity and shorten the analysis time; the purity of the analyte and the test accuracy are effectively improved, and all components and contents in rhizosphere secretion can be qualitatively and quantitatively analyzed; the method can effectively realize the analysis of complex plant rhizosphere secretion, and has good technical advantages when detecting some trace and unknown secretion.
Description
Technical Field
The invention belongs to the fields of analytical chemistry and environmental engineering, and particularly relates to a method for testing plant rhizosphere secretion based on combination of thin film solid phase microextraction (TFME) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS).
Background
The plant root system can release various substances to the soil environment around the root axis in the growth process, namely the plant rhizosphere secretion. The plant rhizosphere secretion has complex composition and low content, the biochemical property is easy to change, and the plant rhizosphere secretion is sensitive to the action of soil microorganisms and the response of soil environmental behaviors, so the plant rhizosphere secretion has great technical difficulty in extraction, separation and identification. The existing measurement methods include: the methods of solvent extraction, resin method, solid phase root zone extraction, gas chromatography and liquid chromatography are combined, and the methods have good extraction capability, but have the following defects: (1) a large amount of samples and organic reagents are needed, the operation process is complicated, and the time is long; (2) the complex operation can cause lower recovery rate and high precision, and the accurate qualitative and quantitative determination of each component and content of the substance to be detected is difficult; (3) interfering substances are easily generated in the extraction process, so that separated components are not real, and further research is influenced. Therefore, on the premise of not damaging plant rhizosphere components and properties, the related methodology bottleneck still needs to be broken through to effectively extract, separate and identify secretion components.
The thin film solid phase microextraction (TFME) method is one of the solid phase microextraction modes, has the characteristics of larger effective extraction phase volume, shorter pre-extraction equilibrium time, larger adsorption capacity and the like, and can quickly reach the final phase equilibrium stage by increasing the mass transfer speed with an analyte. Meanwhile, the TFME technology can be used together with various instruments such as liquid chromatography, gas chromatography, mass spectrum, Raman spectrum and the like, so that the effective extraction volume and the sensitivity and selectivity of analysis and detection are increased. The technology has unique advantages in the aspects of analyzing complex component systems and detecting unknown and trace substances.
The important characteristics of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry are high mass resolution (high mass resolution) and high mass accuracy (high mass accuracy), so that the FT-ICR MS can know the composition of compounds from a molecular level, thereby performing qualitative analysis on known target compounds and unknown compounds, and being very suitable for molecular level characterization of complex matrix samples. TF-ICR MS represents the highest level of MS analysis technology so far, and the resolution of Bruker 9.4T Apex-Ultra FT-ICR MS can reach over 100 ten thousand. The FT-ICR MS can be directly used for sample injection analysis, can reduce the interference of the external environment, simplifies and refines the analysis process, and is very suitable for the analysis and characterization of complex matrix samples such as environment, biology and the like.
Disclosure of Invention
The invention aims to provide a method for determining plant rhizosphere secretion based on combination of thin film solid phase microextraction (TFME) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) by optimizing secretion extraction mode and mass spectrum conditions aiming at the problems that plant rhizosphere secretion is complex in component, trace in amount and greatly disturbed by environment.
In order to realize the purpose, the invention firstly carries out indoor soil pot culture on the sample plant to reach different growth and development stages of the plant, and then samples and analyzes the rhizosphere sample.
The invention relates to a method for measuring plant rhizosphere secretion based on thin film solid phase microextraction-FT-ICR MS combination, which comprises the following steps:
step 1, collecting plant rhizosphere secretion
Digging out the plants when the plants grow to different growth and development stages such as seedlings, strong seedlings, blossoms and withers, washing the plants clean by deionized water, placing the plants in a Hoagland culture solution which is irradiated by an ultraviolet lamp for sterilization and added with 0.05 percent of PPM bacteriostatic agent for water culture; collecting culture solution every 24h, and continuously collecting for many times to obtain aqueous solution of rhizosphere secretion; placing the collected aqueous solution of the rhizosphere secretion in a brown reagent bottle, and storing at the temperature of 4 ℃;
step 2, extracting plant rhizosphere secretion solution by TFME method
Washing the TFME film with a certain amount of deionized water, drying at room temperature for 10-30min, placing the TFME film in the collected aqueous solution of the rhizosphere secretion, stirring at the rotation speed of 600-1000rpm for 15-25min, taking out, immersing with an elution solvent, shaking for elution for 3-7min, releasing the rhizosphere secretion adsorbed on the film into the elution solvent from the film, then rotatably evaporating the elution solvent at 40-60 ℃ to dryness, and fixing the volume of the residual rhizosphere secretion with dichloromethane for later use;
step 3, FT-ICR MS analysis of plant rhizosphere secretion components
Performing component analysis on the constant volume liquid obtained in the step 2 by adopting FT-ICR MS; wherein the electrospray ion source is an ESI ion source, the flow rate of a sample is set to be 160-300 mu L/h, high-purity nitrogen is used as a drying gas and an atomizing gas, the pressure value is 4kPa, and the flow rate is 1.0-8.0L/min; an acquisition mode: positive/negative ions; the polarization voltage is 3.5-4.5kV, the capillary inlet voltage is 4.5-5.5kV, and the capillary outlet voltage is 280-320V; the cumulative time of the ion source six-stage rod is 0.001s, and the cumulative time of the collision pool is 1.0 s; the collection quality range is 50-1000, the collection point number is 4M, and the scanning spectrogram is overlapped 65-250 times.
The method for measuring the plant rhizosphere secretion based on the combination of the thin film solid phase microextraction-FT-ICR MS comprises the following steps:
the TFME film in the step 2 is a special film for a film solid phase microextraction method, and comprises a PA film and a PDMS film; the elution solvent is a mixed solvent formed by mixing dichloromethane, ether, methanol and water according to the volume ratio of 1:1:1: 1.
The invention has the following advantages:
(1) the method has the advantages of no need of preparing a large amount of sample solution, simple operation process, effectively improved detection sensitivity and shortened analysis time.
(2) Effectively improving the purity of the analyte and the test accuracy, and qualitatively and quantitatively analyzing each component and content in the rhizosphere secretion.
(3) The method can effectively realize the analysis of complex plant rhizosphere secretion, and has good technical advantages when detecting some trace and unknown secretion.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
A method for measuring plant rhizosphere secretion based on thin film solid phase microextraction-FT-ICR MS combination comprises the following specific operation steps:
step 1, collecting plant rhizosphere secretion solution
Collecting rhizosphere secretion of the white clover by adopting a soil culture mode, digging out the white clover when the white clover grows to different growth and development stages such as seedling, strong seedling, flowering and withering, washing the white clover clean by deionized water, and placing the washed white clover into Hoagland culture solution which is prepared by deionized water and is added with 0.05 percent of PPM bacteriostatic agent for water culture; sterilizing the Hoagland culture solution containing the bacteriostatic agent by ultraviolet lamp irradiation; collecting culture solution every 24h, and continuously collecting twice to obtain aqueous solution of rhizosphere secretion; placing the collected aqueous solution of the rhizosphere secretion in a brown reagent bottle, and storing at the temperature of 4 ℃;
step 2, extracting plant rhizosphere secretion collecting liquid by TFME method
Selecting a PDMS membrane with the thickness of 100 mu m as a TFME membrane, cleaning the PDMS membrane with 3mL of deionized water, drying the PDMS membrane for 10min at room temperature, placing the PDMS membrane in 10mL of aqueous solution of rhizosphere secretion, stirring the PDMS membrane for 15min at the rotating speed of 600rpm, taking the PDMS membrane out of the solution, immersing the PDMS membrane in 3mL of elution solvent, oscillating the PDMS membrane for 3min, releasing the rhizosphere secretion adsorbed on the PDMS membrane into the elution solvent from the PDMS membrane, then rotationally evaporating the elution solvent in which the rhizosphere secretion is collected to dryness at 40 ℃, and fixing the volume of the elution solvent with dichloromethane for later use; the elution solvent is dichloromethane in volume ratio: diethyl ether: methanol: water is a mixed solvent prepared from 1:1:1: 1;
step 3, FT-ICR MS analysis of plant rhizosphere secretion components
Performing component analysis on the constant volume liquid obtained in the step 2 by adopting FT-ICR MS; the high resolution mass spectrum adopts a 9.4T Apex-Ultra Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) produced by Bruker company in America, an electrospray ion source is an ESI ion source, the flow rate of a sample is set to be 160 mu L/h, high-purity nitrogen is used as dry gas and atomizing gas, the pressure value is 4kPa, and the flow rate is 1.0L/min; an acquisition mode: positive ions/negative ions, the polarization voltage is 3.5kV, the capillary inlet voltage is 4.5kV, and the capillary outlet voltage is 280V; the cumulative time of the ion source six-stage rod is 0.001s, and the cumulative time of the collision pool is 1.0 s; the collection quality range is 50-1000, the collection point number is 4M, and the scanning spectrogram is overlapped 65 times.
Example 2
A method for measuring plant rhizosphere secretion based on thin film solid phase microextraction-FT-ICR MS combination comprises the following specific operation steps:
step 1, collecting plant rhizosphere secretion solution
Collecting rhizosphere secretion of the yellow flag by adopting a soil culture mode, digging out the yellow flag when the yellow flag grows to different growth and development stages such as seedling, strong seedling, flowering and withering, washing the yellow flag clean by using deionized water, and placing the washed yellow flag into Hoagland culture solution which is prepared by deionized water and added with 0.05 Percent of PPM (PPM) bacteriostatic agent for water culture; sterilizing the Hoagland culture solution containing the bacteriostatic agent by ultraviolet lamp irradiation; collecting culture solution every 24h, and continuously collecting for four times to obtain aqueous solution of rhizosphere secretion; placing the collected aqueous solution of the rhizosphere secretion in a brown reagent bottle, and storing at the temperature of 4 ℃;
step 2, extracting plant rhizosphere secretion collecting liquid by TFME method
Selecting a PDMS membrane with the thickness of 70 mu m as a TFME membrane, washing the PDMS membrane with 5mL of deionized water, drying at room temperature for 20min, placing the PDMS membrane in 15mL of aqueous solution of rhizosphere secretion, stirring at the rotating speed of 800rpm for 20min, taking the membrane out of the solution, immersing the membrane with 4mL of elution solvent, oscillating and eluting for 5min, releasing the rhizosphere secretion adsorbed on the membrane into the elution solvent from the membrane, then rotationally evaporating the elution solvent with the collected rhizosphere secretion at 50 ℃ to dryness, and then fixing the volume with dichloromethane for later use; the elution solvent is dichloromethane in volume ratio: diethyl ether: methanol: water is a mixed solvent prepared from 1:1:1: 1;
step 3, FT-ICR MS analysis of plant rhizosphere secretion components
Performing component analysis on the constant volume liquid obtained in the step 2 by adopting FT-ICR MS; the high-resolution mass spectrum adopts a 9.4T Apex-Ultra Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) produced by Bruker company in America, an electrospray ion source is an ESI ion source, the flow rate of a sample is set to be 230 mu L/h, high-purity nitrogen is used as dry gas and atomizing gas, the pressure value is 4kPa, and the flow rate is 5.0L/min; an acquisition mode: the polarization voltage of the positive ions/negative ions is 4.0kV, the inlet voltage of the capillary tube is 5.0kV, and the outlet voltage of the capillary tube is 300V; the cumulative time of the ion source six-stage rod is 0.001s, and the cumulative time of the collision pool is 1.0 s; the collection quality range is 50-1000, the collection point number is 4M, and the scanning spectrogram is superposed for 150 times.
Example 3
A method for measuring plant rhizosphere secretion based on thin film solid phase microextraction-FT-ICR MS combination comprises the following specific operation steps:
step 1, collecting plant rhizosphere secretion solution
Collecting rhizosphere secretion of canna by adopting a soil culture mode, digging out the canna when the canna grows to different growth and development stages such as seedling, strong seedling, flowering, withering and the like, washing the canna by deionized water, and placing the washed canna in Hoagland culture solution which is prepared by deionized water and is added with 0.05 Percent of PPM (PPM) bacteriostatic agent for water culture; sterilizing the Hoagland culture solution containing the bacteriostatic agent by ultraviolet lamp irradiation; collecting culture solution every 24h, and continuously collecting for one week to obtain aqueous solution of rhizosphere secretion; placing the collected aqueous solution of the rhizosphere secretion in a brown reagent bottle, and storing at the temperature of 4 ℃;
step 2, extracting plant rhizosphere secretion collecting liquid by TFME method
Selecting a PA membrane with the membrane thickness of 70 mu m as a TFME membrane, washing the PA membrane with 4mL of deionized water, drying the PA membrane for 30min at room temperature, placing the PA membrane into 20mL of aqueous solution of rhizosphere secretion, stirring the solution at the rotating speed of 1000rpm for 25min, taking the PA membrane out of the solution, immersing the PA membrane with 5mL of elution solvent, oscillating and eluting the PA membrane for 7min, releasing the rhizosphere secretion adsorbed on the PA membrane into the elution solvent from the PA membrane, then rotationally evaporating the elution solvent with the rhizosphere secretion collected at 60 ℃ to dryness, and fixing the volume of the elution solvent with dichloromethane for later use; the elution solvent is dichloromethane in volume ratio: diethyl ether: methanol: water is a mixed solvent prepared from 1:1:1: 1;
step 3, FT-ICR MS analysis of plant rhizosphere secretion components
Performing component analysis on the constant volume liquid obtained in the step 2 by adopting FT-ICR MS; the high resolution mass spectrum adopts a 9.4T Apex-Ultra Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) produced by Bruker company in America, an electrospray ion source is an ESI ion source, the flow rate of a sample is set to be 300 mu L/h, high-purity nitrogen is used as dry gas and atomizing gas, the pressure value is 4kPa, and the flow rate is 8.0L/min; an acquisition mode: the polarization voltage of the positive ions/negative ions is 4.5kV, the inlet voltage of the capillary tube is 5.5kV, and the outlet voltage of the capillary tube is 320V; the cumulative time of the ion source six-stage rod is 0.001s, and the cumulative time of the collision pool is 1.0 s; the collection quality range is 50-1000, the collection point number is 4M, and the scanning spectrogram is overlapped for 250 times.
Claims (2)
1. A method for measuring plant rhizosphere secretion based on a combination of thin film solid phase microextraction-FT-ICR MS is characterized by comprising the following steps:
step 1, collecting plant rhizosphere secretion
Digging out the plants when the plants grow to different growth and development stages such as seedlings, strong seedlings, blossoms and withers, washing the plants clean by deionized water, placing the plants in a Hoagland culture solution which is irradiated by an ultraviolet lamp for sterilization and added with 0.05 percent of PPM bacteriostatic agent for water culture; collecting culture solution every 24h, and continuously collecting for many times to obtain aqueous solution of rhizosphere secretion; placing the collected aqueous solution of the rhizosphere secretion in a brown reagent bottle, and storing at the temperature of 4 ℃;
step 2, extracting plant rhizosphere secretion solution by TFME method
Washing the TFME film with a certain amount of deionized water, drying at room temperature for 10-30min, placing the TFME film in the collected aqueous solution of the rhizosphere secretion, stirring at the rotation speed of 600-1000rpm for 15-25min, taking out, immersing with an elution solvent, shaking for elution for 3-7min, releasing the rhizosphere secretion adsorbed on the film into the elution solvent from the film, then rotatably evaporating the elution solvent at 40-60 ℃ to dryness, and fixing the volume of the residual rhizosphere secretion with dichloromethane for later use;
step 3, FT-ICR MS analysis of plant rhizosphere secretion components
Performing component analysis on the constant volume liquid obtained in the step 2 by adopting FT-ICR MS; wherein the electrospray ion source is an ESI ion source, the flow rate of a sample is set to be 160-300 mu L/h, high-purity nitrogen is used as a drying gas and an atomizing gas, the pressure value is 4kPa, and the flow rate is 1.0-8.0L/min; an acquisition mode: positive/negative ions; the polarization voltage is 3.5-4.5kV, the capillary inlet voltage is 4.5-5.5kV, and the capillary outlet voltage is 280-320V; the cumulative time of the ion source six-stage rod is 0.001s, and the cumulative time of the collision pool is 1.0 s; the collection quality range is 50-1000, the collection point number is 4M, and the scanning spectrogram is overlapped 65-250 times.
2. The method for measuring plant rhizosphere secretion according to claim 1, wherein the TFME membrane in the step 2 is a special membrane for membrane solid phase microextraction, and comprises PA membrane, PDMS membrane; the elution solvent is a mixed solvent formed by mixing dichloromethane, ether, methanol and water according to the volume ratio of 1:1:1: 1.
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