CN114965825B - Method for detecting bio-organic molecule protoporphyrin IX in marine water environment ecological system - Google Patents
Method for detecting bio-organic molecule protoporphyrin IX in marine water environment ecological system Download PDFInfo
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Abstract
The invention discloses a detection method of a bio-organic molecule protoporphyrin IX in a marine water environment ecosystem, which comprises the steps of firstly pre-filtering a seawater sample to obtain a filtrate, then re-filtering the filtrate by using a microporous filter membrane, and after the filtration is finished, extracting the microporous filter membrane for 20 hours in a dark place by using acetone at the temperature of-20 ℃ to obtain an extract liquor. The extract liquid obtained by the treatment method can be used for quantitative detection of protoporphyrin IX, bacteriochlorophyll, chlorophyll a and pheophytin. The method and the technology of the invention are initiated in the marine field at home and abroad, and are suitable for quickly and accurately quantifying protoporphyrin IX in various marine environments. The invention is beneficial to developing and utilizing protoporphyrin IX compound of marine source, and has great significance for developing and utilizing marine natural products and researching the marine natural products in ecology.
Description
Technical Field
The invention relates to a method for detecting protoporphyrin IX, in particular to a method for detecting bio-organic molecule protoporphyrin IX in a marine water environment ecological system. Belongs to the technical field of organic biomolecule detection.
Background
Protoporphyrin IX is an endogenous fluorescent heterocyclic organic compound and a basic natural organic product, which is widely present in the environment on earth where all aerobic organisms live. Protoporphyrin IX is an important precursor substance, can mediate various special physiological functions such as oxygen transport, storage, photosynthesis, participation in methane metabolism and the like, and is also a common prosthetic group of proteins such as cytochrome, catalase, peroxidase, nitrate reductase and the like. As an important natural product, it is biocompatible and interacts with biomolecules, and thus, it can be used to regulate functions of molecules or cells.
At present, protoporphyrin IX is widely applied in the biomedical field, such as photodynamic therapy, antibacterial and antiviral phototherapy, biosensing, bioimaging, research and development of anti-cancer drugs, interference of biochemical pathways and the like, and is widely applied in a plurality of fields such as analysis industries such as signal indicators and the like, research and development of military products and the like.
The ocean occupies 71 percent of the total area of the world, has abundant biological resources, and particularly, the related ocean natural products are to be developed and utilized by human beings. Protoporphyrin IX is widely present in animal, plant and microbial cells and exerts an irreplaceable physiological function. For example, protoporphyrin ix is the only precursor of chlorophyll, under the action of ATP-chelating enzyme, it chelates magnesium ions and gradually forms chlorophyll, while chlorophyll is the main mediator of photosynthesis of green plants, and mediates various biogeochemical cycle processes such as global energy cycle, carbon cycle, nitrogen cycle, and the like. In addition, the microbial secreted bacteriochlorophyll is also derived from the synthesis of protoporphyrin IX, and the microorganisms are the main contributors to the biodiversity on the earth, and play an important role in driving the biogeochemical cycle of biogenic elements such as carbon, nitrogen and the like and regulating the structure and the function of an ecological system. Therefore, the development of natural products such as protoporphyrin IX from marine environment is the key to the development and application of new products in the future.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting a biological organic molecule protoporphyrin IX in a marine water environment ecological system.
In order to realize the purpose, the invention adopts the following technical scheme:
1. a seawater sample treatment method comprises the steps of firstly pre-filtering a seawater sample to obtain a filtrate, then re-filtering the filtrate by using a microporous filter membrane, and after the re-filtering is finished, extracting the microporous filter membrane for 20 hours in a dark place by using acetone at the temperature of-20 ℃ to obtain an extract liquid.
Preferably, the pre-filtration is performed using a screen cloth with a pore size of 10 μm to remove large-sized zooplankton, phytoplankton and debris trash.
Preferably, the microporous filter membrane is a glass fiber filter membrane with the pore diameter of 0.7 μm. The aperture can effectively intercept particle plankton in the seawater environment and is as close to the real value of the environment as possible.
Preferably, the diameter of the microporous membrane is 47mm, and after the filtration is finished, the microporous membrane is completely immersed in 5mL of acetone for extraction in the dark. The microfiltration membrane after filtration will remain 0.8mL water, so the total extraction volume is 5.8mL, acetone final volume concentration of 86%.
2. The treatment method is applied to quantitative detection of protoporphyrin IX, bacteriochlorophyll, chlorophyll a and pheophytin in an ecological system of an ocean water environment.
3. The detection method for the bio-organic molecule protoporphyrin IX in the marine water environment ecological system based on the treatment method comprises the following specific steps:
(1) Firstly, treating a seawater sample by using the treatment method to obtain an extract liquor;
(2) Adjusting the pH of the extract to be less than 2, acidifying the extract at room temperature in a dark place for 24 hours, and realizing the quantitative detection of protoporphyrin IX by utilizing a reversed phase high performance liquid chromatography-mass spectrometry method.
Preferably, in the step (2), pH adjustment is performed by using a 1.2mol/L hydrochloric acid solution.
Preferably, in the step (2), the amount of the sample to be subjected to the reversed-phase high-performance liquid chromatography is in the range of 10 to 100. Mu.L. The range changes according to the change of the sample environment so as to meet the requirement of protoporphyrin IX detection in special environments such as oligotrophic sea area and the like.
Preferably, in the step (2), the chromatographic column adopted by the reversed-phase high performance liquid chromatography is a C18 reversed-phase bonded silica gel column, the column temperature is 34-36 ℃, and the flow rate of the mobile phase is 1mL/min.
Preferably, in step (2), the reversed-phase high performance liquid chromatography uses a fluorescence detector with an excitation wavelength of 406nm and an emission wavelength of 635nm.
Preferably, in the step (2), the mobile phase composition of the reversed phase high performance liquid chromatography comprises the following components in percentage by volume: mobile phase A:60% acetonitrile +39.9% water +0.1% formic acid, mobile phase B:99.9% acetone +0.1% formic acid; the gradient elution procedure was: 20% mobile phase B in 0-2 min, mobile phase B ratio increased from 20% gradient to 100% in 2-2.2 min, 100% mobile phase B hold in 2-10 min, mobile phase B ratio gradient decreased to 20% in 10-10.2 min, 20% mobile phase B hold in 10.2-12 min, the whole gradient program kept mobile phase ratio a + B =100%.
Preferably, in step (2), the mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the scanning mode is a positive ion mode, the temperature of the ion source is 250 ℃, the voltage of a capillary tube is 3KV, the voltage of a nozzle is 1.5KV, the atomizing air pressure is 30psi, the temperature of a drying air is 350 ℃, and the flow rate of the drying air is 11L/min.
Preferably, in step (2), the parent ion has 563m/z, the daughter ions have 504m/z,489m/z and 445m/z, respectively, and the corresponding collision energies are 46eV,55eV and 56eV, respectively, upon mass spectrometry.
The invention has the beneficial effects that: the invention provides a method for treating a seawater sample, which comprises the steps of firstly pre-filtering the seawater sample to obtain a filtrate, then re-filtering the filtrate by using a microporous filter membrane, and after the filtration is finished, extracting the microporous filter membrane for 20 hours in a dark place by using acetone at the temperature of-20 ℃ to obtain an extract. The extract liquid obtained by the treatment method can be used for quantitative detection of protoporphyrin IX, bacteriochlorophyll, chlorophyll a and pheophytin. The method and the technology of the invention are initiated in the marine field at home and abroad, and are suitable for quickly and accurately quantifying protoporphyrin IX in various marine environments. The invention is beneficial to developing and utilizing protoporphyrin IX compound of marine source, and has great significance for developing and utilizing marine natural products and researching the marine natural products in ecology.
The method has the advantages of wide sample source, simple and efficient extraction method, concise and rapid quantitative method, low detection limit, high recovery rate and good stability, can realize batch detection of the samples in a shorter time, and is more suitable for quantitative detection of bio-organic molecule protoporphyrin IX in a marine ecosystem due to the perfection of the detection method. The acetone organic reagent used in the invention can realize cell disruption of an environmental sample and dissolution of a target object to a great extent, can better eliminate signal interference caused by other substances in HPLC detection, can effectively protect the molecular structures of metalized protoporphyrin IX, chlorophyll and bacteriochlorophyll from being damaged, and can reduce the real proportion of environmental pigments to a great extent. The concept and method of the present invention can be extended to the detection and quantification of marine natural products with the same properties or characteristics, such as the detection of porphyrin-based compounds.
The invention greatly improves the extraction effect of protoporphyrin IX by using an efficient extraction technology, and the extraction method is suitable for most of natural water environments. The method can realize effective separation and quantification of the extracting solution by combining with the high performance liquid chromatography, is favorable for obtaining, separating, recovering and purifying the natural protoporphyrin IX compound, can greatly improve the accuracy and precision of a detection result by utilizing the high performance liquid chromatography, and is convenient for obtaining real and reliable data. Is beneficial to disclosing the ecological function of protoporphyrin IX in a marine environment ecosystem and has great research significance for exploring unknown biochemical processes in the global biogeochemical cycle. Particularly, reversed-phase high performance liquid chromatography is selected, and a gradient elution procedure is adopted, so that separation and quantification of protoporphyrin IX in a complex environment can be well realized.
The method firstly uses acetone to extract protoporphyrin IX in the natural environment, specifically uses acetone to break cells, promotes organic molecules to be dissolved or suspended in the acetone, and then realizes the substitution of protons for metal ions in the metallized protoporphyrin IX molecules through acidification treatment, so as to realize the total extraction of free and metallized protoporphyrin IX in the environmental cells, and the free and metallized protoporphyrin IX is in a single-molecule state, thereby accelerating the dissolution of protoporphyrin IX.
Drawings
FIG. 1 is a flow chart of the detection of a biological organic molecule protoporphyrin IX in a marine water environment according to an embodiment of the invention;
FIG. 2 is a full wavelength scan of a protoporphyrin IX standard in an example of the present invention;
FIG. 3 is a reversed phase high performance liquid chromatography separation chart of a protoporphyrin IX standard according to an embodiment of the present invention;
FIG. 4 is a diagram of a reversed phase high performance liquid chromatography separation of a seawater sample according to an embodiment of the present invention;
FIG. 5 is a graph of IX standard curve of protoporphyrin calculated by reference to an embodiment of the present invention;
FIG. 6 is a chart showing the identification of protoporphyrin IX by mass spectrometry in a seawater sample according to an embodiment of the present invention;
FIG. 7 is a graph showing the comparison of the extraction effects of protoporphyrin IX with different reagents in a seawater sample according to an embodiment of the present invention;
FIG. 8 is a comparison of the chromatograms of the separation effect of protoporphyrin IX standard with different flow phases in the examples of the present invention;
FIG. 9 is a comparison of the chromatographic peak areas of a standard protoporphyrin IX with different flow phase separation effects in examples of the present invention;
FIG. 10 is a comparison graph of pH range optimized chromatogram of protoporphyrin IX in a seawater sample according to an embodiment of the present invention;
FIG. 11 is a graph showing the comparison of the acidification time kinetics of protoporphyrin IX in a seawater sample according to an embodiment of the present invention;
FIG. 12 is a graph comparing the concentration of protoporphyrin IX in seawater samples at different locations in accordance with one embodiment of the present invention;
FIG. 13 is a plot of a linear fit of protoporphyrin IX to chlorophyll correlation in a seawater sample according to an embodiment of the present invention;
FIG. 14 is a plot of a linear fit of protoporphyrin IX to bacteriochlorophyll correlation in a seawater sample in accordance with an embodiment of the present invention;
FIG. 15 is a linear fit graph of the correlation between protoporphyrin IX and pheophytin in a seawater sample according to an embodiment of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.
The seawater samples related to the embodiment of the invention are from the water areas of Jiulongjiang, xiamen city, fujian province, china, and the acquisition time of the sample of the embodiment is 11 months in 2021.
Example 1:
a method for detecting a bio-organic molecule protoporphyrin IX in a marine water environment ecosystem refers to a flow chart of FIG. 1 for detecting the bio-organic molecule protoporphyrin IX in the marine water environment, which includes the following steps:
1. sample collection and pretreatment
1000mL of seawater sample was collected on site, prefiltered with a clean sieve of 10 μm pore size to remove large size zooplankton, phytoplankton and debris trash, and the filtrate was stored in a clean polyethylene bottle.
2. Sample filtration and preservation
And (3) carrying out vacuum filtration on the pre-filtered sample according to the quality condition of water, recording the final filtration volume of a glass cellulose filter membrane with the diameter of 47mm and the aperture of 0.7 mu m, folding the filter membrane in half, placing the folded filter membrane in a 5mL centrifuge tube, storing the folded filter membrane in a dark place at the temperature of minus 20 ℃, returning the folded filter membrane to a laboratory, and extracting the sample as far as possible within one month to prevent the sample from being degraded due to long-time placement.
3. Extraction and acidification of samples
Under the condition of darkness as far as possible, transferring the sample into a 15mL centrifuge tube, adding 5mL of acetone with the purity of 100% (about 0.8mL of water remains after filtering through a 47mm GF/F filter membrane, so that the total volume of extraction is 5.8mL, the actual reaction concentration of the acetone is 86%, namely the extraction is easier to realize by using an acetone aqueous solution with the final volume concentration of 86%), slightly shaking and uniformly mixing to ensure that the filter membrane is completely immersed in the acetone, and extracting for 20 hours at-20 ℃ in a dark place.
After extraction, the filter membrane was pressed to the bottom of the tube with clean forceps, and the supernatant was transferred to a 5mL centrifuge tube as much as possible and stored at-20 ℃ in the dark for future use. And sucking 500 mu L of filtrate, adding 1.2mol/L hydrochloric acid prepared by 100 mu L of deionized water (namely, the sample is diluted by 1.2 times), pressing and filtering by using a 0.45 mu m organic filter membrane and a 1mL sterile syringe, uniformly mixing, acidifying at room temperature in a dark place for 24 hours, and detecting protoporphyrin IX. The acetone extract filtered by a filter membrane of 0.45 mu m can be directly used for the bacteriochlorophyll detection. The untreated acetone extract can be directly used for fluorescence quantitative detection of chlorophyll a and pheophytin (acidification is carried out for about 30s by adding 1-2 drops of hydrochloric acid).
4. Reversed phase high performance liquid chromatography
Separating and identifying the extracted protoporphyrin IX-containing extract liquor by reverse phase high performance liquid chromatography, wherein the specific operating conditions are as follows:
high performance liquid chromatography column types: c18 reverse phase bonded silica gel column, chromatographic column pH tolerance range of 1-10 (Shimadzu InertSustain C18, 4.6X 150mm,5 μm);
the mobile phase composition is as follows: mobile phase A:60% acetonitrile +39.9% water +0.1% formic acid, mobile phase B:99.9% acetone +0.1% formic acid;
gradient elution procedure: 20% mobile phase B in 0-2 min, mobile phase B proportion increased from 20% gradient to 100% in 2-2.2 min, 100% mobile phase B hold in 2-10 min, 20% mobile phase B proportion gradient decreased to 20% in 10-10.2 min, 20% mobile phase B hold in 10.2-12 min, mobile phase proportion a + B =100% for the entire gradient program, and total run time for the system program was 12 min.
The temperature of the chromatographic column is 35 plus or minus 1 ℃, the temperature of the liquid phase automatic sample injection plate is 4 plus or minus 2 ℃, the sample injection volume is 20-100 mu L, and an illuminating lamp prepared by the sample injector needs to be closed during automatic sample injection so as to prevent the sample from volatilizing due to the heat of the lamp.
The excitation wavelength of a fluorescence detector is 406nm and the emission wavelength is 635nm during chromatographic detection.
5. Liquid chromatography-mass spectrometry combined method for identifying protoporphyrin IX compound in seawater sample
Mass spectrometry: an Agilent 6490 triple tandem quadrupole LC MS equipped with an Agilent 1290Infinity LC system and Agilent MassHunter data processing software;
and (3) chromatographic column: c18 reverse phase bonded silica gel column, column pH tolerance range of 1-10 (Shimadzu Inertsustain C18, 4.6X 150mm,5 μm)
Mobile phase composition: a mobile phase A:60% acetonitrile +39.9% water +0.1% formic acid, mobile phase B:99.9% acetone +0.1% formic acid;
gradient elution procedure: 20% mobile phase B in 0-2 min, mobile phase B proportion increased from 20% gradient to 100% in 2-2.2 min, 100% mobile phase B hold in 2-10 min, 20% mobile phase B proportion gradient decreased to 20% in 10-10.2 min, 20% mobile phase B hold in 10.2-12 min, mobile phase proportion a + B =100% for the entire gradient program, and total run time for the system program was 12 min.
The temperature of the chromatographic column is 35 plus or minus 1 ℃, the temperature of the liquid phase automatic sample injection plate is 4 plus or minus 2 ℃, the sample injection volume is 10 mu L, and the flow rate is 0.7 mL/min.
An ion source: electrospray ion source
Scanning mode: positive ion mode
Capillary voltage: 3KV
Ion source temperature: 250 ℃ C
Nozzle voltage: 1.5KV
Pressing an atomizer: 30psi
Temperature of the drying gas: 350 deg.C
Flow rate of drying gas: 11L/min
Scanning range: 480-620m/z
The compound is identified by LC-MS/MS mass spectrum scanning by adopting the set conditions, and the method comprises the following steps:
scanning in a positive ion mode according to the substance molecular weight (562.6 g/mol) of protoporphyrin IX to determine that the parent ion of the protoporphyrin IX compound is 563m/z, the first-order daughter ion is 504m/z, and the optimal collision energy is 46eV; the secondary ion is 489m/z, and the optimal collision energy is 55eV; the tertiary ion is 445m/z, and the optimal collision energy is 56.
And determining the chromatographic peak retention time of the target protoporphyrin IX according to the chromatogram, and drawing a standard curve by taking the concentration of the protoporphyrin IX standard as a horizontal coordinate and the chromatographic peak area of the standard as a vertical coordinate to obtain a corresponding linear regression equation. And carrying out quantitative analysis on the protoporphyrin IX of the seawater sample according to the peak area obtained by sample detection and a standard curve.
FIG. 2 is a full wavelength scanning of a protoporphyrin IX standard in one embodiment of the present invention, which is effective for fluorescence excitation of protoporphyrin IX in a seawater sample with 406nm wavelength as excitation light.
FIG. 3 is a reversed phase high performance liquid chromatography separation chart of protoporphyrin IX standard in the example of the present invention, under the set liquid chromatography conditions, the effective separation of protoporphyrin IX standard and chlorophyll standard can be realized.
FIG. 4 is a reversed phase high performance liquid chromatography separation chart of a seawater sample according to an embodiment of the present invention, in which the seawater sample can also achieve a better separation effect with other compounds with reference to the retention time of protoporphyrin IX standard, and the retention time of the sample ranges from 4.5 to 5.2 minutes.
FIG. 5 is a graph of a standard curve of protoporphyrin IX calculated by reference to an example of the present invention, with the abscissa as the concentration of the standard protoporphyrin IX and the ordinate as the area of the response peak, and a linear regression equation of y =2995.8869x +79.6585, where R is 2 =0.9989, can be effectively applied to the quantitative detection of protoporphyrin IX in a seawater sample.
FIG. 6 is a liquid chromatography-mass spectrometry identification chart of protoporphyrin IX in a seawater sample, which directly illustrates that the extract contains target organic molecule protoporphyrin IX, and the target peak can be qualitatively determined to be protoporphyrin IX compound by combining the results of liquid chromatography and mass spectrometry.
Example 2:
referring to fig. 1, a flow chart of detecting a bio-organic molecule protoporphyrin ix in a marine water environment according to an embodiment of the present invention, the following conditions are optimized for an extracted seawater sample:
1. extraction reagent optimization
The method comprises the steps of respectively carrying out dark cold extraction on a seawater sample under the same condition by using methanol, ethanol, acetonitrile and acetone as extracting agents, and respectively carrying out acidification and non-acidification chromatographic detection on each extract by using acetone/acetonitrile as a mobile phase. As shown in FIG. 7, the extraction of protoporphyrin IX by acidified acetone was significantly better than that by methanol, ethanol and acetonitrile, and the acidification was more effective than the non-acidified extraction. The research reports at home and abroad show that protoporphyrin IX is very slightly soluble in water, and can be dissolved in most organic solvents and acidic or alkaline aqueous solutions. Although methanol can also extract porphyrin compounds, against the original intention of the present invention, methanol causes isomerization and trans-esterification of chlorophyll upon extraction of chlorophyll, resulting in changes in the molecular structure of chlorophyll, and extraction efficiency of acetone is less than 100%, and thus is not suitable for the present invention. The acetone is used for extracting a plurality of target pigment samples, including chlorophyll, bacteriochlorophyll and pheophytin, by a one-step acetone extraction method, so that the aim of one-step multi-purpose is really fulfilled, the one-step acetone extraction treatment is only needed for one-time extraction of one sample, and the multi-purpose acetone extraction solution can be simultaneously used for detecting protoporphyrin IX, chlorophyll a, pheophytin and bacteriochlorophyll a. The method of one-step multiple use can greatly reduce the concentration distribution proportion among various pigments in the real environment on the premise of protecting the original structure of the pigment molecules from being damaged, avoids concentration difference caused by different treatments or samples, and is more suitable for ecological environment research. The subsequent acidification step of protoporphyrin IX can realize the proton substitution process of metalized protoporphyrin IX, the extraction of free and metalized protoporphyrin IX in environmental cells is realized as far as possible, and the obtained total protoporphyrin IX can better reflect the correlation with other biological pigments, so that the method has extremely important ecological and statistical significance for the function research of protoporphyrin IX in an ecological system. Therefore, although the extraction effect is better when acetone + hydrochloric acid is used as an extracting agent, the method is not suitable for the concept of 'one-step multiple use', and sample-to-sample errors are caused when the acetone + hydrochloric acid is compared with pigment molecules such as chlorophyll, so that the method is not suitable for environmental ecology research.
2. Mobile phase optimization
The protoporphyrin IX standard prepared from formic acid is subjected to HPLC detection by respectively taking methanol/formic acid, ethanol/formic acid, acetonitrile/formic acid, acetone and acetone/acetonitrile/formic acid as mobile phases so as to optimize the separation effect of different organic solvents on protoporphyrin IX. As shown in fig. 8 and fig. 9, the chromatographic peak heights of the mobile phases are, from large to small, acetone/acetonitrile/formic acid > acetone > ethanol/formic acid > methanol/formic acid > acetonitrile/formic acid, and the chromatographic peak smoothing effect using acetone/acetonitrile/formic acid as the mobile phase is significantly better than that of other mobile phases in terms of the degree of separation. The chromatographic peak area result shows that the peak area of the mobile phase containing acetone/formic acid is obviously higher than that of the other three organic solvents, wherein the separation effect of acetonitrile/formic acid on protoporphyrin IX is weakest. Therefore, the selection of acetone/formic acid with high elution capacity and acetonitrile/formic acid with low efficiency as mobile phases can ensure that the target substance is separated by acetone as efficiently as possible and the non-target substance is separated by acetonitrile, and the combination of the high and low elution phases is more suitable for the invention.
3. Sample pH range optimization
Using hydrochloric acid as acidity regulator and sodium hydroxide as alkalinity regulator, respectively adding 500 μ L of seawater sample extractive solution into 6 brown sample bottles of 1.5mL, respectively regulating pH value to 1,3,5 with hydrochloric acid; the pH was adjusted to about 9 and 11 with sodium hydroxide, and the sample without any acid or base was used as a neutral sample (pH = 7), and after mixing uniformly, the reaction was carried out for 24 hours in the dark.
Fig. 10 is a comparison graph of pH range optimized chromatography of protoporphyrin ix in a seawater sample according to an embodiment of the present invention, and according to a liquid chromatography detection result, it can be clearly seen that pH has a great influence on the separation effect of protoporphyrin ix in the seawater sample, and when pH =1, a target shows a good separation degree, and when pH >3, the sample shows a multi-peak or hetero-peak state, and effective separation and quantification cannot be completed. The results also demonstrate that acidic organic solvents are beneficial to the dissolution and chromatographic separation of protoporphyrin IX single molecules.
4. Sample acidification time optimization
The pH range optimization results of the samples showed that pH had a large effect on the degree of separation, so we performed kinetic monitoring of the acidification time of the samples based on low acidic conditions. The temperature of an automatic sample feeding disc of the high performance liquid chromatograph is set to be room temperature, the same acidified sample is continuously monitored for 24 hours at intervals of 1 hour, and 5 parallel samples are arranged in each group of samples. And the monitoring result takes acidification time as an abscissa, and the sample concentration change is plotted as an ordinate to draw a trend graph.
The results are shown in fig. 11, which is a comparison graph of the kinetics of the acidification time of protoporphyrin ix in a seawater sample according to the present invention, and it can be seen that the concentration of protoporphyrin ix produced by the acidification of the sample gradually increases with the continuous extension of the acidification time, and the graph in fig. 11 shows chromatograms of 0 hour, 12 hours and 24 hours respectively for the acidification time, and it can be clearly seen that the chromatographic peak changes significantly with the extension of the acidification time. The rising concentration of protoporphyrin IX may be due to the difference in the rate of replacement of metal ions by hydrogen ions. Comparing the IX concentration change of protoporphyrin in the last 3 hours, the result shows that there is no significant difference in 3 concentrations, therefore, based on the consideration of timeliness and practical application, it is recommended that the acidification time is uniformly established to be 24 hours.
Example 3:
the methodology established by the method of the invention was verified according to the detection methods of example 1 and example 2, and the specific contents are as follows:
1. detection limit and quantification limit
A0.2 nmol/L protoporphyrin IX standard was injected into the liquid chromatography for determination of detection limit and quantitation limit, containing 6 replicates. Defining the average peak area of protoporphyrin IX as a response signal, calculating the signal-to-noise ratio by taking the relative standard deviation of the peak areas of 6 parallel samples as background noise, defining the detection limit as the concentration of protoporphyrin IX corresponding to 3 times of the signal-to-noise ratio, and defining the concentration of protoporphyrin IX corresponding to 10 times of the signal-to-noise ratio as the quantitative limit.
The detection result shows that the signal-to-noise ratio is 3.8, the detection limit is 3.83 +/-1.00 pM, and the quantification limit is 12.77 +/-3.34 pM, which shows that the method has extremely low detection limit and quantification limit, and can be suitable for detecting and quantifying protoporphyrin IX in a water environment with low environmental content.
2. Accuracy, precision and recovery validation
In order to verify the accuracy and precision of sample detection among different batches within the same day and within different days, 4 concentration gradients of protoporphyrin IX were selected for verification. The IX concentration gradients of protoporphyrin were set at 0.5nM,1nM,5nM and 60nM, respectively. The intra-day detection interval is often 6 hours and the extra-day interval is 4 days. And (3) selecting protoporphyrin IX with the standard substance concentration of 1nM/mL for recovery rate determination, recovering the standard substance according to the steps described in example 1, performing recovery rate verification according to the steps described in example 2, and expressing the recovery rate according to the ratio of the detection value to the true value.
The results are shown in table 1, with average accuracy between 80-95% and accuracy error between 0.3-14% for different concentration levels over the day; the extraterrestrial accuracy is between 90 and 99 percent, and the accuracy error is between 2 and 4.6 percent, which indicates that the detection of the instrument is relatively accurate. The average recovery rate range is 97.48 +/-1.92%, and the recovery efficiency is high.
TABLE 1 verification of accuracy and precision of protoporphyrin IX in seawater samples according to examples of the present invention
Example 4:
according to the detection methods of example 1 and example 2, quantitative detection of protoporphyrin IX was carried out on a sample in an actual environment. Samples were collected at 11 months in 2021 at the time of maritime voyage survey in the water area of jiulongjiang, mansion, fujian province and the gulf sea area of mansion, 0.5-1 m surface water sample and 1000mL bottom water sample were collected on site, and the samples were collected and processed as described in the examples. Quantitative and qualitative detection of protoporphyrin IX in seawater samples was performed as described in example 2.
The results of the tests are shown in fig. 12 and table 2, which show that the concentration of protoporphyrin ix changes with the salinity of seawater, specifically, the concentration of protoporphyrin with lower salinity at the upstream of jiulongjiang is higher than that of the sample with high salinity at the bay of mansion, and the content of protoporphyrin ix in the surface sample is higher than that in the bottom sample at the upstream of jiulongjiang, but on the contrary, the contents of protoporphyrin ix in the bay of mansion tend to be consistent.
Table 2 shows the result of detecting protoporphyrin IX in a seawater sample from Jiulongjiang-Xiamenwan in the examples of the present invention
Since protoporphyrin IX is a direct precursor of chlorophyll and bacteriochlorophyll, we comparatively analyzed the relationship between protoporphyrin IX and chlorophyll, bacteriochlorophyll and pheophytin under the same environment. FIGS. 13, 14 and 15 are graphs of linear fits of the correlation between protoporphyrin IX and chlorophyll a, bacteriochlorophyll a and pheophytin, respectively, and the surface layer linear fits R between chlorophyll, bacteriochlorophyll and pheophytin in the examples of the present invention 2 0.8946, 0.8476 and 0.9061 respectively, linear fit R of bottom protoporphyrin IX to bottom chlorophyll, bacteriochlorophyll and pheophytin 2 0.6949, 0.6244 and 0.8108 respectively, which shows that protoporphyrin IX in natural seawater samples has a great positive correlation with its derivatives chlorophyll, bacteriochlorophyll and pheophytin, and provides reliable data and technical support for the application of protoporphyrin IX organic biomolecules in ecology.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, the scope of the present invention is not limited thereto, and various modifications and changes that can be made by those skilled in the art without inventive efforts based on the technical solutions of the present invention are within the scope of the present invention.
Claims (4)
1. A method for detecting bio-organic molecule protoporphyrin IX in a marine water environment ecosystem is characterized by comprising the following specific steps:
(1) Firstly, pre-filtering a seawater sample to obtain a filtrate, then, re-filtering the filtrate by using a microporous filter membrane, and after the re-filtering is finished, performing light-resistant extraction on the microporous filter membrane for 20 hours by using acetone at the temperature of-20 ℃ to obtain an extract liquid;
(2) Adjusting the pH of the extract to be less than 2, acidifying the extract at room temperature in a dark place for 24 hours, and realizing quantitative detection on protoporphyrin IX by using a reversed-phase high performance liquid chromatography-mass spectrometry method;
in the step (2), a chromatographic column adopted by the reversed-phase high-performance liquid chromatography is a C18 reversed-phase bonded silica gel column, the column temperature is 34-36 ℃, and the flow rate of a mobile phase is 1mL/min;
the reversed-phase high performance liquid chromatography adopts a fluorescence detector, the excitation wavelength of the fluorescence detector is 406nm, and the emission wavelength of the fluorescence detector is 635 nm;
the mobile phase composition of the reversed phase high performance liquid chromatography comprises the following components in percentage by volume: mobile phase A:60% acetonitrile +39.9% water +0.1% formic acid, mobile phase B:99.9% acetone +0.1% formic acid; the gradient elution procedure was: 20% of mobile phase B in 0-2 min, the proportion of the mobile phase B is increased from 20% to 100% in 2-2.2 min, 100% of the mobile phase B is maintained in 2-10 min, the proportion of the mobile phase B is decreased to 20% in 10-10.2 min, 20% of the mobile phase B is maintained in 10.2-12 min, and the proportion of the mobile phase A + B =100% in the whole gradient program;
the mass spectrometry conditions were as follows: the ion source is an electrospray ion source, the scanning mode is a positive ion mode, the temperature of the ion source is 250 ℃, the voltage of a capillary tube is 3KV, the voltage of a nozzle is 1.5KV, the atomizing air pressure is 30psi, the temperature of a drying air is 350 ℃, and the flow rate of the drying air is 11L/min.
2. The detection method according to claim 1, wherein prefiltering is performed using a bolting silk with a pore size of 10 μm to remove large-sized zooplankton, phytoplankton and debris garbage.
3. The detection method according to claim 1, wherein the microfiltration membrane is a glass fiber filtration membrane having a pore size of 0.7 μm.
4. The detection method according to claim 1, wherein the diameter of the microporous filter membrane is 47mm, and after the filtration is completed, the microporous filter membrane is completely immersed in 5mL of acetone and extracted away from light.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057194A (en) * | 1988-12-12 | 1991-12-25 | Fmc公司 | The preparation of porphyrin and application |
| JP2008089358A (en) * | 2006-09-29 | 2008-04-17 | Bl:Kk | Immunological detection method and device of protoporphyrins in biological sample |
| BR102014021479A2 (en) * | 2014-08-29 | 2016-08-23 | Univ São Paulo Usp | process of obtaining biocompatible polymeric micro and nanocapsules, biocompatible polymeric micro and nanocapsules and their use as antimalarial |
-
2022
- 2022-05-21 CN CN202210558220.0A patent/CN114965825B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057194A (en) * | 1988-12-12 | 1991-12-25 | Fmc公司 | The preparation of porphyrin and application |
| JP2008089358A (en) * | 2006-09-29 | 2008-04-17 | Bl:Kk | Immunological detection method and device of protoporphyrins in biological sample |
| BR102014021479A2 (en) * | 2014-08-29 | 2016-08-23 | Univ São Paulo Usp | process of obtaining biocompatible polymeric micro and nanocapsules, biocompatible polymeric micro and nanocapsules and their use as antimalarial |
Non-Patent Citations (5)
| Title |
|---|
| Heme in the marine environment: from cells to the iron cycle;Shane L Hogle 等;《Metallomics》;20140422;第6卷(第6期);第1107-1120页 * |
| Intrinsic Fluorescence of Protoporphyrin IX from Blood Samples Can Yield Information on the Growth of Prostate Tumours;Flávia Rodrigues de Oliveira Silva 等;《Journal of Fluorescence》;20100424;第20卷;第1159-1165页 * |
| 分光光度法测定海水中叶绿素含量的研究;金霞 等;《广州化工》;20100430;第38卷(第4期);第132-136页 * |
| 南黄海夏末叶绿素α的分布特征;李宝华 等;《海洋与湖沼》;19990531;第30卷(第3期);第300-305页 * |
| 海水胁迫对菠菜(Spinacia olerancea L.)叶绿体活性氧和叶绿素代谢的影响;孙锦 等;《生态学报》;20090831;第29卷(第8期);第4361-4371页 * |
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