CN109765202B - Method for rapidly detecting bacterial endotoxin - Google Patents
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- CN109765202B CN109765202B CN201910073945.9A CN201910073945A CN109765202B CN 109765202 B CN109765202 B CN 109765202B CN 201910073945 A CN201910073945 A CN 201910073945A CN 109765202 B CN109765202 B CN 109765202B
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Abstract
The invention provides a method for rapidly detecting bacterial endotoxin based on surface-assisted laser desorption ionization mass spectrometry. According to the method, the zinc ferrite chitosan composite material is prepared and used as a matrix, so that a method for rapidly detecting bacterial endotoxin based on an organic mass spectrometry technology is developed, the coffee ring effect of the traditional organic matrix is overcome, and the rapid detection of bacterial endotoxin is facilitated. The method has the advantages of no need of complex sample pretreatment, small sample consumption (0.1 mu L), simple sample preparation, less time consumption, good salt tolerance, good reproducibility and the like, and has good application prospect in the field of food and medicine safety.
Description
Technical Field
The invention relates to a method for rapidly detecting bacterial endotoxin based on surface-assisted laser desorption ionization mass spectrometry, in particular to application of the method in medicine and food detection.
Background
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is a novel high-efficiency soft ionization organic mass spectrometry, which is very simple and high-efficiency theoretically or in design, and has become one of indispensable tools for biomacromolecule analysis in recent years. The instrument mainly comprises two parts: matrix-assisted laser desorption ionization ion source (MALDI) and time-of-flight mass analyzer (TOF). The principle of MALDI is to irradiate a co-crystallized thin film formed by a sample and a matrix with laser light, where the energy absorbed by the matrix is transferred to the sample molecules, causing the sample to be analyzed to vaporize and ionize. The principle of TOF is that ions are accelerated to fly through a flight tube under the action of an electric field, and are detected according to different flight times of arriving at a detector, namely, the mass-to-charge ratio (M/Z) of the ions is measured to be in direct proportion to the flight time of the ions, and the ions are detected. But the biggest limitation is that the traditional matrix and the sample have non-uniform phenomenon when being co-crystallized, thereby seriously influencing the repeatability of the experiment. The surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) developed based on MALDI-MS well solves the problem of reproducibility and has the advantage of high-throughput rapid screening.
Endotoxin (Endotoxin) is a unique structure of a cell wall of gram-negative bacteria, and the main component of the Endotoxin is hydrophilic Lipopolysaccharide (LPS), and the Endotoxin is released only after the bacteria die and dissolve or the bacterial cells are destroyed by an artificial method, and is an exogenous pyrogen. Endotoxin is widely present in air, soil, water and other environments closely related to human life, a pyrogen entering a blood circulation system of a human body can cause a series of adverse reactions such as fever, and the human body is very sensitive to the immune reaction of the endotoxin, and trace (1-5 ng/kg body weight) of the endotoxin can cause the body temperature to rise, thereby causing serious problems such as fever reaction, leucocyte reaction, microcirculation disturbance, endotoxin shock and the like. Therefore, in the aspects of public health and food and drug inspection systems and the like, the detection of endotoxin in various medicines, foods, beverages, water sources and biological products is strengthened, the content of endotoxin is strictly controlled and limited below an acceptable legal level, and the public safety of the health and social health of people can be guaranteed.
At present, the detection method of endotoxin mainly comprises a Rabbit Pyrogen method (Rabbit Pyrogen Test), a Limulus Test (LT), a gas chromatography-mass spectrometry combined method, a spectral technology analysis and the like. The rabbit pyrogen method is the earliest method for detecting endotoxin, but the method generally wastes time and labor, has more interference factors, has poor reproducibility of a detection result and the like, and thus the requirement of detection is difficult to meet. The limulus test method is a gold standard for endotoxin detection, is the most sensitive bacterial endotoxin detection method at present, and still has the defects of harsh reaction conditions, numerous influencing factors, difficult acquisition of limulus sources and the like. In recent years, endotoxin detection methods based on spectroscopic techniques have overcome the above disadvantages, but still have the disadvantages of large sample consumption, the need for complicated instrumentation, difficulty in qualitative and quantitative determination, and the like. The chromatography-mass spectrometry combined technology has both high-efficiency chromatographic separation performance and ultra-strong qualitative and quantitative mass spectrometry capacity, is an ideal analysis technology, and is widely applied to the detection of microorganisms and toxins at present. Nevertheless, efficient extraction or enrichment of endotoxins from complex samples will remain the biggest bottleneck faced by chromatography-mass spectrometry techniques. In conclusion, the existing analytical detection methods have some defects or shortcomings, so that the development of a high-sensitivity, simple, high-throughput and rapid endotoxin detection method is imperative.
Disclosure of Invention
Aiming at the defects, the invention provides a method for rapidly detecting bacterial endotoxin based on surface-assisted laser desorption ionization mass spectrometry, which develops a method for rapidly detecting bacterial endotoxin based on an organic mass spectrometry technology by preparing a zinc-ferrite chitosan composite material and using the zinc-ferrite chitosan composite material as a matrix, overcomes the coffee ring effect of the traditional organic matrix, is beneficial to rapidly detecting bacterial endotoxin, and performs qualitative analysis on the bacterial endotoxin. Meanwhile, the used sample amount is very small (0.1 mu L), the detection speed is high, complex sample pretreatment is not needed, and the method has high application value when being used for the actual detection of endotoxin in food and drugs.
The technical scheme provided by the invention is as follows:
a method for rapidly screening and detecting bacterial endotoxin based on surface-assisted laser desorption ionization mass spectrometry comprises the following steps:
step 1: preparation of reagents and instruments: a laser desorption ionization time-of-flight mass spectrometer, a bacterial endotoxin working standard, a zinc ferrite chitosan composite material, an injection and drinking water;
step 2: preparing a zinc ferrite chitosan composite material matrix;
and step 3: mixing the zinc ferrite chitosan composite material matrix and a bacterial endotoxin working standard substance on a target plate, and drying at room temperature;
and 4, step 4: and carrying out mass spectrum detection, and analyzing the result to obtain a conclusion.
Further, the preparation of the zinc ferrite chitosan composite material comprises the following steps:
step 1: preparing zinc ferrite: weighing zinc oxide, sodium acetate and FeCl3·6H2O and Na3Cit·2H2O was dissolved in ethylene glycol and the resulting solution was transferred to a reaction kettle. The reaction kettle is placed in an oven with the temperature of 210 ℃ of 170-. Washing the obtained magnetic product with water and ethanol for 3-5 times respectively, and drying at room temperature, wherein zinc oxide, sodium acetate and FeCl are added3·6H2O and Na3Cit·2H2The mass ratio of O is 1: (7-9): (3-5): (0.01-0.03);
step 2: preparing a zinc ferrite chitosan composite material: adding zinc ferrite and chitosan into acetic acid solvent, and stirring at constant temperature of 50-70 ℃ for 1-3 h. The product was isolated using a magnet and washed with water and ethanol to near neutrality. And (3) drying the cleaned product in vacuum at 40-60 ℃ to obtain the zinc ferrite chitosan composite material.
The mass ratio of the zinc ferrite to the chitosan is 1 (2-4).
Further, the food and medicine comprise Vc injection and drinking water.
The invention has the following advantages:
1) the prepared substrate has low cost and is easy to synthesize;
2) the prepared matrix has good reproducibility and wide quality detection range;
3) the dosage of the detected food and medicine is very small (0.1 mu L), and the detection process is simple;
4) the analysis speed is high (less than 20 minutes), and the information is visual;
5) the prepared matrix can be as low as 0.1EU/mL when being used for detecting bacterial endotoxin standard substances;
6) the prepared matrix is a nano material, has good energy transfer capacity, can not form crystals after natural drying, and overcomes the coffee ring effect of the traditional organic matrix after drying.
Drawings
Fig. 1 is a scanning electron microscope image of a zinc ferrite chitosan composite material prepared in example 2 of the present invention;
FIG. 2 is a transmission electron microscope image of the zinc ferrite chitosan composite material prepared in example 2 of the present invention;
FIG. 3 is a mass spectrum detection result of a bacterial endotoxin working standard substance by using a zinc ferrite chitosan composite material as a matrix;
FIG. 4 is a mass spectrum detection result of Vc injection liquid with zinc ferrite chitosan composite material as a matrix;
FIG. 5 shows the mass spectrometric detection results of drinking water using zinc ferrite chitosan composite as matrix.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.
Example 1
The preparation method of the zinc ferrite chitosan composite material matrix comprises the following steps:
step 1: preparing zinc ferrite: weighing zinc oxide, sodium acetate and FeCl3·6H2O and Na3Cit·2H2O was dissolved in ethylene glycol and the resulting solution was transferred to a reaction kettle. The reaction kettle is placed in an oven at 200 ℃ for reaction for 12 hours. Washing the obtained magnetic product with water and ethanol for 4 times respectively, and drying at room temperature, wherein zinc oxide, sodium acetate and FeCl are added3·6H2O and Na3Cit·2H2The mass ratio of O is 1: 7: 3: 0.01;
step 2: preparing a zinc ferrite chitosan composite material: adding zinc ferrite and chitosan into an acetic acid solvent, and stirring for 2h at a constant temperature of 60 ℃. The product was isolated using a magnet and washed with water and ethanol to near neutrality. And (3) drying the cleaned product in vacuum at 50 ℃ to obtain the zinc ferrite chitosan composite material.
The mass ratio of the zinc ferrite to the chitosan is 1: 2.
Example 2
The preparation method of the zinc ferrite chitosan composite material matrix comprises the following steps:
step 1: preparing zinc ferrite: weighing zinc oxide, sodium acetate and FeCl3·6H2O and Na3Cit·2H2O was dissolved in ethylene glycol and the resulting solution was transferred to a reaction kettle. The reaction kettle is placed in an oven at 200 ℃ for reaction for 12 hours. Washing the obtained magnetic product with water and ethanol for 4 times respectively, and drying at room temperature, wherein zinc oxide, sodium acetate and FeCl are added3·6H2O and Na3Cit·2H2The mass ratio of O is 1: 8: 4: 0.03;
step 2: preparing a zinc ferrite chitosan composite material: adding zinc ferrite and chitosan into an acetic acid solvent, and stirring for 2h at a constant temperature of 60 ℃. The product was isolated using a magnet and washed with water and ethanol to near neutrality. And (3) drying the cleaned product in vacuum at 50 ℃ to obtain the zinc ferrite chitosan composite material.
The mass ratio of the zinc ferrite to the chitosan is 1: 3.
Wherein, the embodiment 2 is the best embodiment for preparing the zinc ferrite chitosan composite material matrix.
Characterization of the matrix:
the instruments used for characterization were: tecnai G220 Transmission Electron Microscopy (TEM); s-4800 Scanning Electron Microscope (SEM).
The characterization result is as follows:
the zinc ferrite chitosan composite material is approximately spherical through SEM images, and a plurality of nanoclusters exist on the surface. The TEM image shows that the zinc ferrite chitosan composite material has uniform particle size, and the average particle size is about 220 nm.
Example 3
The preparation method of the zinc ferrite chitosan composite material matrix comprises the following steps:
step 1: preparing zinc ferrite: weighing zinc oxide, sodium acetate and FeCl3·6H2O and Na3Cit·2H2O was dissolved in ethylene glycol and the resulting solution was transferred to a reaction kettle. The reaction kettle is placed in an oven at 200 ℃ for reaction for 12 hours. Washing the obtained magnetic product with water and ethanol for 4 times respectively, and drying at room temperature, wherein zinc oxide, sodium acetate and FeCl are added3·6H2O and Na3Cit·2H2The mass ratio of O is 1: 9: 5: 0.02;
step 2: preparing a zinc ferrite chitosan composite material: adding zinc ferrite and chitosan into an acetic acid solvent, and stirring for 2h at a constant temperature of 60 ℃. The product was isolated using a magnet and washed with water and ethanol to near neutrality. And (3) drying the cleaned product in vacuum at 50 ℃ to obtain the zinc ferrite chitosan composite material.
The mass ratio of the zinc ferrite to the chitosan is 1: 4.
Example 4: working standard substance for detecting bacterial endotoxin by using zinc ferrite chitosan composite material as matrix
(1) Preparing an instrument: laser desorption ionization time-of-flight mass spectrometer; detection is performed in a linear mode, positive ion mode detection.
(2) Mixing the prepared zinc ferrite chitosan composite material with a bacterial endotoxin working standard substance, placing the mixture on a target plate, and drying at room temperature;
(3) and (5) carrying out mass spectrum detection.
The mass spectrum results are shown in FIG. 3, in which the polymer peak around m/z 7000 is the characteristic peak of bacterial endotoxin.
Example 5: vc injection with zinc ferrite chitosan composite material as matrix
(1) Preparing an instrument: laser desorption ionization time-of-flight mass spectrometer; detection is performed in a linear mode, positive ion mode detection.
(2) Mixing the prepared zinc ferrite chitosan composite material with the Vc injection, dotting the mixture on a target plate, and drying at room temperature;
(3) and (5) carrying out mass spectrum detection.
The mass spectrum results are shown in fig. 4, and the characteristic peak of the Vc injection without bacterial endotoxin is measured.
Example 6: zinc ferrite chitosan composite material is used as drinking water
(1) Preparing an instrument: laser desorption ionization time-of-flight mass spectrometer; detection is performed in a linear mode, positive ion mode detection.
(2) Mixing the prepared zinc ferrite chitosan composite material with drinking water, putting the mixture on a target plate, and drying at room temperature;
(3) and (5) carrying out mass spectrum detection.
The mass spectrometry results are shown in fig. 5, and the measured drinking water is free of characteristic peaks of bacterial endotoxin.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (4)
1. A method for rapidly detecting bacterial endotoxin based on surface-assisted laser desorption ionization mass spectrometry is characterized in that a zinc-ferrite chitosan composite material matrix and a sample to be detected are mixed and placed on a target plate, and mass spectrometry detection is carried out by utilizing a laser desorption ionization time-of-flight mass spectrometer and is used for rapidly detecting bacterial endotoxin;
the preparation method of the zinc ferrite chitosan composite material comprises the following steps:
1) preparing zinc ferrite: weighing zinc oxide, sodium acetate and FeCl3·6H2O and Na3Cit·2H2Dissolving O in ethylene glycol, transferring the obtained solution to a reaction kettle, and placing the reaction kettle at 170-2Reacting for 10-14 hours in a drying oven at 10 ℃, washing the obtained magnetic product with water and ethanol, and drying at room temperature;
2) preparing a zinc ferrite chitosan composite material: adding zinc ferrite and chitosan into an acetic acid solvent, stirring for 1-3h at a constant temperature of 50-70 ℃, separating a product by using a magnet, washing the product to be nearly neutral by using water and ethanol, and drying the cleaned product in vacuum at 40-60 ℃ to obtain the zinc ferrite chitosan composite material.
2. The method for rapidly detecting bacterial endotoxin based on surface-assisted laser desorption ionization mass spectrometry as claimed in claim 1, wherein zinc oxide, sodium acetate and FeCl used in preparation of zinc ferrite3·6H2O and Na3Cit·2H2The mass ratio of O is 1: (7-9): (3-5): (0.01-0.03).
3. The method for rapidly detecting bacterial endotoxin based on surface-assisted laser desorption ionization mass spectrometry as claimed in claim 1, wherein the mass ratio of zinc ferrite to chitosan used in preparing the zinc ferrite chitosan composite material is 1 (2-4).
4. The method for rapidly detecting bacterial endotoxin based on surface-assisted laser desorption ionization mass spectrometry (SARPS-MS) of claim 1, wherein the amount of the sample to be detected is 0.1 μ L.
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| WO2015135071A1 (en) * | 2014-03-14 | 2015-09-17 | Hancock Robert E W | Diagnostic for sepsis |
| CN105435745A (en) * | 2015-08-20 | 2016-03-30 | 浙江师范大学 | Zinc ferrite@collagen/graphene oxide composite material and preparation method therefor and application thereof |
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| WO2015135071A1 (en) * | 2014-03-14 | 2015-09-17 | Hancock Robert E W | Diagnostic for sepsis |
| CN105435745A (en) * | 2015-08-20 | 2016-03-30 | 浙江师范大学 | Zinc ferrite@collagen/graphene oxide composite material and preparation method therefor and application thereof |
| CN107807185A (en) * | 2017-10-20 | 2018-03-16 | 中国科学院武汉物理与数学研究所 | A kind of LC-MS analysis endotoxin lipoid A method |
| CN109030616A (en) * | 2018-07-19 | 2018-12-18 | 福州大学 | A method of utilizing surface assisted laser desorption ionization mass spectrum rapid screening and identification edible oil |
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