CN115337670B - Functionalized solid-phase microextraction probe and application thereof in live detection of tetrodotoxin - Google Patents
Functionalized solid-phase microextraction probe and application thereof in live detection of tetrodotoxin Download PDFInfo
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- CN115337670B CN115337670B CN202210809169.6A CN202210809169A CN115337670B CN 115337670 B CN115337670 B CN 115337670B CN 202210809169 A CN202210809169 A CN 202210809169A CN 115337670 B CN115337670 B CN 115337670B
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- 239000000523 sample Substances 0.000 title claims abstract description 160
- CFMYXEVWODSLAX-QOZOJKKESA-N tetrodotoxin Chemical compound O([C@@]([C@H]1O)(O)O[C@H]2[C@@]3(O)CO)[C@H]3[C@@H](O)[C@]11[C@H]2[C@@H](O)N=C(N)N1 CFMYXEVWODSLAX-QOZOJKKESA-N 0.000 title claims abstract description 84
- 229950010357 tetrodotoxin Drugs 0.000 title claims abstract description 84
- CFMYXEVWODSLAX-UHFFFAOYSA-N tetrodotoxin Natural products C12C(O)NC(=N)NC2(C2O)C(O)C3C(CO)(O)C1OC2(O)O3 CFMYXEVWODSLAX-UHFFFAOYSA-N 0.000 title claims abstract description 84
- 238000002470 solid-phase micro-extraction Methods 0.000 title claims abstract description 54
- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 229920002239 polyacrylonitrile Polymers 0.000 claims abstract description 86
- 241000251468 Actinopterygii Species 0.000 claims abstract description 56
- 239000011248 coating agent Substances 0.000 claims abstract description 44
- 238000000576 coating method Methods 0.000 claims abstract description 44
- 229910001220 stainless steel Inorganic materials 0.000 claims abstract description 43
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- 238000000034 method Methods 0.000 claims abstract description 34
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- 238000000605 extraction Methods 0.000 claims abstract description 28
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- 239000003053 toxin Substances 0.000 claims abstract description 9
- 231100000765 toxin Toxicity 0.000 claims abstract description 9
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 claims abstract description 7
- 229960002748 norepinephrine Drugs 0.000 claims abstract description 7
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- 230000000379 polymerizing effect Effects 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 68
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 36
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 31
- 238000003795 desorption Methods 0.000 claims description 29
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- 239000012071 phase Substances 0.000 claims description 20
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- 229910021641 deionized water Inorganic materials 0.000 claims description 16
- RRAFCDWBNXTKKO-UHFFFAOYSA-N eugenol Chemical compound COC1=CC(CC=C)=CC=C1O RRAFCDWBNXTKKO-UHFFFAOYSA-N 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 16
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- NPBVQXIMTZKSBA-UHFFFAOYSA-N Chavibetol Natural products COC1=CC=C(CC=C)C=C1O NPBVQXIMTZKSBA-UHFFFAOYSA-N 0.000 claims description 8
- 239000005770 Eugenol Substances 0.000 claims description 8
- UVMRYBDEERADNV-UHFFFAOYSA-N Pseudoeugenol Natural products COC1=CC(C(C)=C)=CC=C1O UVMRYBDEERADNV-UHFFFAOYSA-N 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 229960002217 eugenol Drugs 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 206010002091 Anaesthesia Diseases 0.000 claims description 7
- 241001441723 Takifugu Species 0.000 claims description 7
- 241001627955 Tetraodon lineatus Species 0.000 claims description 7
- 238000001727 in vivo Methods 0.000 claims description 7
- 210000003205 muscle Anatomy 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
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- 239000008399 tap water Substances 0.000 claims description 2
- 235000020679 tap water Nutrition 0.000 claims description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims 3
- 239000002131 composite material Substances 0.000 claims 1
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- 238000007654 immersion Methods 0.000 description 10
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/265—Adsorption chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N2030/042—Standards
- G01N2030/047—Standards external
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention discloses a functionalized solid-phase microextraction probe and application thereof in living body detection of tetrodotoxin, wherein the probe is obtained by coating graphene oxide-polyacrylonitrile oxide solution on an activated stainless steel needle and spontaneously polymerizing PNE coating on the surface, one end of the activated stainless steel needle is vertically immersed in the graphene oxide-polyacrylonitrile oxide solution, the activated stainless steel needle is slowly taken out and heated to fix the coating, the immersing and heating steps are repeated to uniformly wrap the coating on the activated stainless steel needle, and the activated stainless steel needle is immersed in norepinephrine solution to spontaneously polymerize the surface of the activated stainless steel needle to form the coating. The functionalized solid-phase microextraction probe can detect the toxin content of the puffer fish in the living puffer fish, the detection limit meets the safety eating requirement of the puffer fish, the detection does not need to kill the puffer fish, the extraction and detection processes can be completed within 1 hour, the sensitivity is high, the detection cost is low, and the sample lethality problem caused by detecting the toxin of the puffer fish is effectively avoided.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a functional solid-phase microextraction probe and application thereof in the living body detection of tetrodotoxin.
Background
The edible history of the globefish in China is long, and the globefish is a large country for producing and selling the globefish, and has rich globefish resources. However, the tetrodotoxin is easily affected by various pollutants in the environment in the growth process of the tetrodotoxin, so that the tetrodotoxin is accumulated in the body, serious threat is generated to the eating safety of the tetrodotoxin, and people can die when serious threat is generated. Moreover, the tetrodotoxin has high stability, is difficult to destroy by a common cooking method, and has no targeted antidote or antitoxin after poisoning. Therefore, it is important to detect the toxin of the puffer fish by a scientific and effective method, so as to avoid the toxic event of the puffer fish. The existing methods for detecting the tetrodotoxin comprise a mouse biological method, an immunoassay method, an instrumental analysis method and the like, and have some defects. For example, chinese patent application (publication No. CN 201410603308.5) discloses "method for determining tetrodotoxin in marine organisms by immunoaffinity column purification-liquid chromatography-tandem mass spectrometry", which uses unique selective recognition of immunoaffinity column and high sensitivity and accuracy of liquid chromatography-tandem mass spectrometry to determine tetrodotoxin in marine organisms, but is disadvantageous in that: in the pretreatment process, the tetrodotoxin is extracted by using a solvent and purified by using an immunoaffinity column, the steps are complicated, the immunoaffinity column can be prepared and completed after more than ten days, the time is relatively consumed, and the detection process is lethal to the sample. The Chinese patent application (publication number: CN 201410251894.1) discloses a tetrodotoxin detection kit which can rapidly and simply detect tetrodotoxin by using an immune enhanced latex turbidimetry method, and realizes mass detection, but has the following defects: the determination after the sample is dissolved and homogenized destroys the higher economic value of the puffer fish, and the preparation process of the nano emulsion particles combined with the monoclonal antibody of the puffer fish toxin is also more complicated. Therefore, it is necessary to find a rapid and simple living detection method for tetrodotoxin.
The Solid Phase Microextraction (SPME) technology is a relatively novel solvent-free extraction method, integrates collection, separation and enrichment, and is a convenient, quick and environment-friendly sample pretreatment technology. SPME has less effect on the whole sample system and can be used to detect or analyze target components in living animals. For example, chinese patent application (publication No. CN 112547030A) discloses a super-crosslinked polymer nanoparticle solid-phase microextraction biocompatible probe and application thereof in living body analysis, and quartz fiber containing super-crosslinked polymer nanoparticles and a poly-norepinephrine biocompatible coating is used as a solid-phase microextraction probe for pesticide residue analysis in a plant living body, and the preparation process of the probe is complicated. Based on the advantages of SPME technology in living body detection, a living body detection method for the tetrodotoxin is urgently needed to be constructed to explore the residual level of the tetrodotoxin in a fish body, and no relevant report on the living body detection of the tetrodotoxin based on a solid phase microextraction technology exists in the prior art.
Disclosure of Invention
Aiming at the defects of long preparation time, high detection cost and sample lethality in the detection of the tetrodotoxin in the prior art, the invention mainly aims to provide a functionalized solid-phase microextraction probe which has high sensitivity and low cost.
The invention also aims to provide a preparation method of the functionalized solid-phase microextraction probe, which is prepared by an impregnation method.
The invention also aims to provide the application of the functionalized solid phase microextraction probe in the live detection of the tetrodotoxin, and the functionalized solid phase microextraction probe has short preparation time, high sensitivity and low detection cost, and effectively avoids sample death caused by detecting the tetrodotoxin
Sex problems.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a functionalized solid phase microextraction probe obtained by coating an activated stainless steel needle with a graphene oxide-polyacrylonitrile solution and spontaneously polymerizing the norepinephrine solution on the surface thereof to form a PNE coating
Wherein:
the GO-PAN solution is prepared by dissolving Graphene Oxide (GO) in Polyacrylonitrile (PAN) glue, and the preparation method comprises the following steps: mixing PAN solid and N, N-Dimethylformamide (DMF) at a mass ratio of 1:7-8, and stirring thoroughly to obtain PAN fraction
Uniformly dispersing and properly dissolving to form a viscous suspension, heating at 80-100 ℃ for 50-70 min to enable PAN to be completely dissolved in DMF to form yellow clear transparent PAN glue, and cooling to room temperature; adding GO powder into DMF to be dispersed evenly by ultrasonic,
the mass volume ratio of the two is 15-25mg:200-300 mu L of the obtained GO solution is added into the PAN glue and stirred uniformly;
the activated stainless steel needle is obtained by cutting a medical stainless steel needle with the diameter of 0.4-0.6 mm into small sections with the length of 2-3 cm, sequentially carrying out ultrasonic pretreatment in concentrated hydrochloric acid for 8-12 min, taking out and carrying out surface activation treatment of deionized water rinsing.
In a second aspect, the present invention provides a method for preparing the functionalized solid phase microextraction probe, which is prepared by an immersion method, and includes: one end of the activated stainless steel needle is vertically immersed into the GO-PAN solution, and is slowly taken out at 80-100 DEG C
Heating for 2-4min until DMF is fully volatilized to fix the coating, repeating the above steps of immersing and heating for several times to uniformly wrap the coating on the activated stainless steel needle, immersing into the NE solution to spontaneously polymerize 24 h on the surface of the NE solution, taking out,
washing with deionized water to obtain the product; the concentration of the NE solution was 0.2mg/mL, and the NE solution was dissolved by stirring in a mixed solution (1:1, v/v) of 10 mM of a Tri buffer solution having a pH of 8.5 and methanol.
Preferably, the surface coating thickness of the functionalized solid phase microextraction probe is 50-70 μm.
In a third aspect, the invention provides the application of the functionalized solid phase microextraction probe in the living body detection of the tetrodotoxin.
Preferably, the detection limit of the functionalized solid phase microextraction probe on the tetrodotoxin in the tetrodotoxin muscle is 32 ng/g, the quantitative limit is 150 ng/g and the linear range is 50-1000 ng/g.
Preferably, the method for detecting the tetrodotoxin living body by the functionalized solid-phase microextraction probe comprises the following steps:
(1) Anaesthetizing live globefish with eugenol;
(2) A small hole with a depth of 1.5 cm is pricked on the side surface of the back muscle near the fin by a medical injector needle;
(3) Extracting the medical injector needle from the small hole, and inserting the functional solid phase microextraction probe into the small hole for living body extraction;
(4) Fishing out the puffer fish from water, and taking out the functionalized solid-phase microextraction probe after re-anesthesia;
(5) Washing the functionalized solid phase microextraction probe with deionized water, and then desorbing the extracted tetrodotoxin with a desorption solvent;
(6) And (5) carrying out LC-MS/MS analysis on the desorption liquid in the step (5) to obtain the toxin content of the puffer fish in the fish body.
Preferably, in the steps (1) and (4), the method for anaesthetizing the puffer fish comprises the following steps: adding 5 mL eugenol liquid into 5L tap water to obtain 0.1% eugenol (volume fraction) water solution, taking out the puffer fish, and soaking in the water solution
In the liquid, the fish is fished out immediately after the body of the fish loses balance after a few seconds, and anesthesia is completed.
Preferably, in the step (3), the time of the living body extraction is 30-70 min.
Preferably, in the step (5), the desorption solvent is selected from methanol, 50% methanol, acetonitrile and 50% acetonitrile, and the desorption time is 20-40 min.
Preferably, in step (6), the LC-MS/MS analysis conditions are: ultra performance liquid chromatography-triple quadrupole mass spectrometer (Nexera LC30AD & SCIEX SelexION Triple Quad)
5500 System), the mass spectrum ion source was electrospray ion source (ESI), and HILIC column (2.1X100 mm,1.7 μm) of Agilent technologies Co., ltd., U.S. was used for separation, and mobile phase A was aqueous solution containing 0.1% formic acid, flow
The mobile phase B is acetonitrile with the concentration of 0.1%, the flow rate is 0.4mL/min, and the column temperature is 40 ℃;
the gradient elution procedure was as follows: starting with 5% mobile phase A and 95% mobile phase B, after holding for 0.5min, mobile phase A rises to 60% in 2.5min, then rises to 95% in 1min, then remains unchanged for 2min, and then falls back in 0.1min
To 5%, maintaining for 1.9min, and analyzing for 8min for each sample;
the mass spectrometry conditions were as follows: adopting ESI positive ion mode, gas curtain gas of 35psi, ionization voltage of 5500V, desolvation temperature of 500 ℃, atomization gas of 55psi, auxiliary heating gas of 55psi, and Multiple Reaction Monitoring (MRM) for sample alignment
The product was analyzed.
Preferably, in the step (6), the method for quantifying the concentration of the tetrodotoxin in the desorption liquid by adopting an external standard method comprises the following steps: dissolving tetrodotoxin in small amount of 1% acetic acid water solution (v/v), and fixing volume with methanol to obtain concentrations
0.1, 0.5, 1, 5, 10, 50, 100 ng/mL standard gradient solution of tetrodotoxin, using LC-MS/MS to analyze the standard gradient solution of tetrodotoxin, using the concentration as x axis and the peak area as y axis, drawing the standard curve of tetrodotoxin
And (3) quantifying the concentration of the tetrodotoxin in the desorption liquid.
Compared with the prior art, the invention has the beneficial effects that:
(1) The functionalized solid-phase microextraction probe can detect the toxin content of the puffer fish in the living puffer fish, the detection limit can meet the requirement of safe eating of the puffer fish, and the preparation method is simple and convenient, high in sensitivity and low in cost.
(2) The functionalized solid-phase microextraction probe is used for detecting the tetrodotoxin in the living tetrodotoxin fish, does not need to kill the tetrodotoxin fish, has shorter detection time, can complete the extraction and detection processes within 1 hour, and has high sensitivity and detection cost
The method has the advantages of being low in cost, effectively avoiding the sample lethality problem caused by detecting the tetrodotoxin, protecting the high economic value of the tetrodotoxin, and being more in line with the ethics of animal experiments.
Drawings
FIG. 1 is a photograph of a GO-PAN@PNE probe in an example.
FIG. 2 is a scanning electron micrograph of a GO-PAN@PNE probe in an example.
FIG. 3 is a standard curve obtained by LC-MS/MS analysis of tetrodotoxin standards in the examples.
FIG. 4 is a LC-MS/MS chromatogram obtained by extracting the tetrodotoxin with the labeled fish meat by the GO-PAN@PNE probe in the example.
FIG. 5 is the results of extraction of 1. Mu.g/g of tetrodotoxin with 1. Mu.g/mL of labeled fish and 1. Mu.g/mL of aqueous solution, respectively, with a blank PAN probe, a GO-PAN probe and a GO-PAN@PNE probe.
FIG. 6 is an optimization of the extraction time of GO-PAN@PNE probe extracted tetrodotoxin added fish meat in the example.
FIG. 7 is a desorption solvent optimization of GO-PAN@PNE probe extraction of the tetrodotoxin-labeled fish meat in the example.
FIG. 8 is a graph showing the optimization of the desorption time of the GO-PAN@PNE probe in the example for the extraction of the tetrodotoxin and the labeled fish meat.
FIG. 9 is a linear range of GO-PAN@PNE probe extraction of tetrodotoxin plus fish in the examples.
FIG. 10 is a photograph showing the detection of tetrodotoxin in tetrodotofish by the GO-PAN@PNE probe in the example.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited to the following scope. The reagents used in the examples, which were not noted, were commercially available and conventional products.
The solid phase microextraction (SPME technique) based method for detecting tetrodotoxin in vivo in the following examples comprises the following steps:
(1) Preparation of GO-PAN solution: mixing PAN solid and DMF with the mass ratio of 1:7-1:8, and stirring thoroughly to disperse PAN uniformly and dissolve properly to form a viscous suspension. Heating in an oven at 80-100deg.C for 50-70 min to obtain a mixture
PAN was completely dissolved in DMF to make a yellow clear transparent PAN gum, which was cooled to room temperature. Adding 15-25mg of GO powder into 200-300 mu L of DMF, and uniformly dispersing by ultrasonic treatment 1-h. GO solution to be uniformly dispersed
Adding the PAN glue, and uniformly stirring to prepare the GO-PAN solution.
(2) The GO-PAN@PNE probe is prepared by an immersion method: cutting medical stainless steel needle with diameter of 0.4-0.6. 0.6 mm into small segments with length of 2-3 cm, ultrasonic pre-treating in concentrated hydrochloric acid for 8-12 min, taking out, and rinsing with deionized water to obtain the final product
And (5) activating the surface of the medical stainless steel needle. Vertically immersing one end of the treated medical stainless steel needle with a length of about 1cm into the solution prepared in the step (1), slowly taking out, heating at 80-100deg.C for 2-4min to volatilize DMF,
the coating is fixed. The immersing and heating steps were repeated several times to make the coating as uniformly wrapped as possible on the medical stainless steel needle and to make the final coating thickness about 50-70 μm. NE was dissolved in Tri-buffer (10 mM, pH=8.5) and
in a mixed solution of methanol (1:1, v/v) (concentration 0.2 mg/mL). The prepared probe was immersed in the NE solution for 24. 24 h to spontaneously polymerize NE on the probe surface. After the time is up, the probe is taken out and repeatedly rinsed with deionized water, namely
Obtaining the GO-PAN@PNE probe.
(3) Living body detection of tetrodotoxin Using GO-PAN@PNE probe: the puffer fish is fished out of the water, and immersed in 0.1% eugenol (volume fraction) aqueous solution for anesthesia until the body of the puffer fish is out of balance. Fishing out the fish to make
A small hole of about 1.5 a cm a depth was made in the dorsal muscle side near the fin with a medical syringe needle. The syringe was pulled out of the well and then a self-made probe was inserted into the well. In the sampling process, the fugu obscurus is put back into clear water
Extracting for 30-70 min. After extraction, the fish is again fished out of the water, and after anesthesia, the probe is removed. After washing the probes 2 to 4 s with pure water, desorption was performed using a desorption solvent.
(4) Instrument analysis: and (3) detecting the desorption liquid obtained in the step (3) through LC-MS/MS, and determining the toxin content level of the fugu fish in the fugu fish body by adopting an external standard method.
In some embodiments, in step (3), the specific method parameters for in vivo detection of tetrodotoxin using the self-made probe are: the self-made probe has extraction time of 30-70 min for tetrodotoxin, and the desorption solvent can be methanol,
50% methanol, acetonitrile and 50% acetonitrile, and the desorption time is 20-40 min.
In some embodiments, in step (4), the detection method of liquid chromatography tandem mass spectrometry comprises: ultra performance liquid chromatography-triple quadrupole mass spectrometer (Nexera LC30AD & SCIEX Selexion) was used
The analysis was performed using a Triple Quad 5500 System) and the mass spectrometry ion source was an electrospray ion source (ESI) and was separated using HILIC column (2.1X100 mm,1.7 μm) from Agilent technologies, inc. of America. Mobile phase A is a mixture containing
0.1% formic acid in water, mobile phase B was 0.1% acetonitrile, flow rate 0.4mL/min, column temperature 40 ℃. The gradient elution procedure was as follows: starting with 5% mobile phase A,95% mobile phase B, after 0.5min, mobile phase A was maintained for 2.5min
To 60%, then to 95% in 1min, again for 2min, then back to 5% in 0.1min, again for 1.9 min. The analysis time for each sample was 8min. The mass spectrometry conditions were as follows: ESI positive ion mode, air curtain gas
35psi ionization voltage 5500V, desolvation temperature 500 ℃, atomizing gas 55psi, auxiliary heating gas 55psi, multiple Reaction Monitoring (MRM) was used to analyze the samples.
In some embodiments, in the step (4), the specific method for quantifying the tetrodotoxin in the desorption solution by using the external standard method comprises the following steps: dissolving tetrodotoxin in small amount of 1% acetic acid water solution (v/v), and fixing volume with methanol
Preparing 0.1, 0.5, 1, 5, 10, 50, 100 ng/mL standard gradient solution of tetrodotoxin, analyzing the standard gradient solution of tetrodotoxin by using an instrument analysis method, drawing with the concentration as x-axis and the peak area as y-axis
And (5) outputting a tetrodotoxin standard curve, and quantifying the tetrodotoxin concentration of the desorption liquid.
The technical scheme of the invention is further explained by the following specific examples.
Example 1
The GO-PAN@PNE probe used for solid phase microextraction is prepared by coating GO-PAN solution on an activated stainless steel needle, and adding a self-polymerized PNE coating on the surface, and can be directly used for in-vivo detection of tetrodotoxin in tetrodotoxin bodies
The content level of the tetrodotoxin in the tetrodotoxin body is shown in the figure 1, and the preparation process comprises the following steps:
(1) Activating the surface of a medical stainless steel needle to prepare GO-PAN solution and NE solution;
(2) Vertically immersing the activated stainless steel needle with the length of about 1cm at one end into the GO-PAN solution, and slowly taking out to finish the immersion;
(3) Placing the impregnated probe into an oven, and heating at 90 ℃ for 3 min until DMF volatilizes to fix the coating;
(4) Repeating the immersing-heating steps in the steps (2) and (3) for a plurality of times, so that the coating is uniformly wrapped on the medical stainless steel needle as much as possible, and the thickness of the final coating is about 50 mu m;
(5) Immersing the prepared probe in the step (4) into a NE solution to spontaneously polymerize the NE on the surface of the probe;
(6) And taking out the probe after 24 and h, and flushing the probe with deionized water to obtain the GO-PAN@PNE solid phase microextraction probe, as shown in a picture of fig. 1.
Example 2
The preparation method of the GO-PAN@PNE probe for solid phase microextraction comprises the following specific steps:
(1) Activating the surface of a medical stainless steel needle to prepare GO-PAN solution and NE solution;
(2) Vertically immersing the activated stainless steel needle with the length of about 1cm at one end into the GO-PAN solution, and slowly taking out to finish the immersion;
(3) Placing the impregnated probe into an oven and heating at 90 ℃ for 3 min until DMF volatilizes, so that the coating is fixed;
(4) Repeating the immersing-heating steps in the steps (2) and (3) for a plurality of times, so that the coating is uniformly wrapped on the medical stainless steel needle as much as possible, and the thickness of the final coating is about 50 mu m;
(5) Immersing the prepared probe in the NE solution in the step (4) to spontaneously polymerize the NE on the surface of the probe;
(6) And taking out the probe after 24 and h, and flushing the probe with deionized water to obtain the GO-PAN@PNE solid phase microextraction probe.
The surface morphology of the solid phase microextraction probe of example 2 GO-PAN@PNE was characterized using SEM. Cutting the probe into a proper size, adhering the probe to a sample stage by using conductive adhesive, and using Nova Nano-SEM 450 type field emission scanning electron
The surface morphology of the probe was observed by a microscope (FESEM, FEI company, usa), and a scanning electron microscope photograph is shown in fig. 2. Under lower magnification, the whole surface of the probe coating presents a smoother and uniform form without larger convex
The uniform distribution of the clusters, depressions and agglomerates is advantageous for improving the reproducibility of the probe. At higher magnification, however, the probe surface was observed to become progressively roughened, giving rise to a more folded and porous structure, because
After DMF in the solution evaporates, the remaining PAN and GO are fixed on the surface of the probe to form an uneven structure, and the structure is favorable for increasing the contact area and improving the load capacity of the probe, thereby improving the extraction performance.
Example 3
The preparation method of the GO-PAN@PNE probe for solid phase microextraction comprises the following specific steps:
(1) Activating the surface of a medical stainless steel needle to prepare GO-PAN solution and NE solution;
(2) Vertically immersing the activated stainless steel needle with the length of about 1cm at one end into the GO-PAN solution, and slowly taking out to finish the immersion;
(3) Placing the impregnated probe into an oven and heating at 90 ℃ for 3 min until DMF volatilizes, so that the coating is fixed;
(4) Repeating the immersing-heating steps in the steps (2) and (3) for a plurality of times, so that the coating is uniformly wrapped on the medical stainless steel needle as much as possible, and the thickness of the final coating is about 50 mu m;
(5) Immersing the prepared probe in the step (4) into a NE solution to spontaneously polymerize the NE on the surface of the probe;
(6) And taking out the probe after 24 and h, and flushing the probe with deionized water to obtain the GO-PAN@PNE solid phase microextraction probe.
The method comprises the following specific steps of extracting the puffer fish flesh by using a GO-PAN@PNE probe: killing and dissecting Fugu obscurus obtained by purchasing several tails, mincing back muscle into meat paste, weighing 5 g-10 mL glass sample bottle,
adding corresponding concentration of tetrodotoxin water solution 5 mL to obtain tetrodotoxin labeled fish sample. The probe was inserted vertically into a 10 mL glass sample bottle containing a sample of labeled fish to ensure complete immersion of the coating in the fish. Extraction knot
After the probe 3 was rinsed with ultrapure water (UpPCL) for 3 s, then dried with a Kimwipe paper towel, the extracted probe was immersed in 250. Mu.L of methanol, and the tetrodotoxin extracted from the probe was desorbed, and the desorption solution was subjected to LC-MS/MS analysis.
The LC-MS/MS instrument analysis conditions were: analysis was performed using an ultra performance liquid chromatography-triple quadrupole mass spectrometer (Nexera LC30AD & SCIEX SelexION Triple Quad 5500 System) from Shimadzu corporation. Quality of the body
The spectral ion source is an electrospray ion source (ESI). The separation was performed using HILIC column (2.1X100 mm,1.7 μm) from Agilent technologies Co., ltd. Mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% acetonitrile
The flow rate was 0.4mL/min and the column temperature was 40 ℃. The gradient elution procedure was as follows: starting with 5% mobile phase A,95% mobile phase B, after 0.5min, mobile phase A rises to 60% in 2.5min, then rises to 95% in 1min, and then remains again
The time period was kept constant for 2min, and then was reduced back to 5% within 0.1min, and maintained for 1.9 min. The analysis time for each sample was 8min. The mass spectrometry conditions were as follows: ESI positive ion mode, air curtain 35psi, ionization voltage 5500V, desolvation temperature
The sample was analyzed using MRM at 500℃with 55psi of atomizing gas and 55psi of auxiliary heating gas.
FIG. 3 is a standard curve obtained by analyzing the standards for tetrodotoxin by the LC-MS/MS method in example 3, which has good curvilinearity, and shows that the LC-MS/MS method can be used for quantitative analysis of tetrodotoxin. FIG. 4 is a view of the use of example 3
LC-MS/MS chromatogram obtained by extracting the fish flesh of the puffer fish with the GO-PAN@PNE probe shows that the probe can extract the toxin of the puffer fish from the fish flesh of the puffer fish.
Example 4
The preparation method of the GO-PAN@PNE probe for solid phase microextraction comprises the following specific steps:
(1) Activating the surface of a medical stainless steel needle to prepare GO-PAN solution and NE solution;
(2) Vertically immersing the activated stainless steel needle with the length of about 1cm at one end into the GO-PAN solution, and slowly taking out to finish the immersion;
(3) Placing the impregnated probe into an oven and heating at 90 ℃ for 3 min until DMF volatilizes, so that the coating is fixed;
(4) Repeating the immersing-heating steps in the steps (2) and (3) for a plurality of times, so that the coating is uniformly wrapped on the medical stainless steel needle as much as possible, and the thickness of the final coating is about 50 mu m;
(5) Immersing the prepared probe in the step (4) into a NE solution to spontaneously polymerize the NE on the surface of the probe;
(6) And taking out the probe after 24 and h, and flushing the probe with deionized water to obtain the GO-PAN@PNE solid phase microextraction probe.
FIG. 5 is a graph showing the results of extraction of 1. Mu.g/g of tetrodotoxin with 1. Mu.g/mL of labeled fish and 1. Mu.g/mL of aqueous solution, respectively, using a blank PAN probe, a GO-PAN probe and a GO-PAN@PNE probe, and the GO-added probe compared to the blank PAN probe
Aiming at the great improvement of the extraction amount of the tetrodotoxin, the GO has an adsorption effect on the tetrodotoxin. When the probe did not encapsulate the PNE, the extraction in the fish with the label was lower than in the aqueous solution, indicating that PNE was conferred
The probe has good anti-biological corrosion capability, and the extraction performance of the probe in fish flesh is improved.
Example 5
The preparation method of the GO-PAN@PNE probe for solid phase microextraction comprises the following specific steps:
(1) Activating the surface of a medical stainless steel needle to prepare GO-PAN solution and NE solution;
(2) Vertically immersing the activated stainless steel needle with the length of about 1cm at one end into the GO-PAN solution, and slowly taking out to finish the immersion;
(3) Placing the impregnated probe into an oven and heating at 90 ℃ for 3 min until DMF volatilizes, so that the coating is fixed;
(4) Repeating the immersing-heating steps in the steps (2) and (3) for a plurality of times, so that the coating is uniformly wrapped on the medical stainless steel needle as much as possible, and the thickness of the final coating is about 50 mu m;
(5) Immersing the prepared probe in the step (4) into a NE solution to spontaneously polymerize the NE on the surface of the probe;
(6) And taking out the probe after 24 and h, and flushing the probe with deionized water to obtain the GO-PAN@PNE solid phase microextraction probe.
Extracting with GO-PAN@PNE probe in fish meat of Takifugu with a weight concentration of 1 μg/g for 5-60 min (5, 10, 15, 20, 30, 45, 60 min), and selecting optimal extraction time
As shown in FIG. 6, the extraction amount increased slowly after 30 min, and 30 min was selected as the optimal extraction time.
Extracting with GO-PAN@PNE probe in fish flesh of Takifugu obscurus with a target concentration of 1 μg/g, desorbing with methanol, acetonitrile, 50% methanol (v: v), 50% acetonitrile (v: v) and ultrapure water as desorption solvents, and selecting the most suitable
As a result of the desorption of the solvent, as shown in fig. 7, the extraction amount was maximized with methanol as the desorption solvent, and methanol was selected as the desorption solvent.
Extracting with GO-PAN@PNE probe in fish flesh of Takifugu with a weight concentration of 1 μg/g, desorbing at time intervals of 10-60 min (10, 15, 20, 30, 45, 60 min), and selecting optimal desorption time
As a result, as shown in FIG. 8, the extraction amount increased slowly after 15 min, and 15 min was selected as the optimal extraction time.
Example 6
The preparation method of the GO-PAN@PNE probe for solid phase microextraction comprises the following specific steps:
(1) Activating the surface of a medical stainless steel needle to prepare GO-PAN solution and NE solution;
(2) Vertically immersing the activated stainless steel needle with the length of about 1cm at one end into the GO-PAN solution, and slowly taking out to finish the immersion;
(3) Placing the impregnated probe into an oven and heating at 90 ℃ for 3 min until DMF volatilizes, so that the coating is fixed;
(4) Repeating the immersing-heating steps in the steps (2) and (3) for a plurality of times, so that the coating is uniformly wrapped on the medical stainless steel needle as much as possible, and the thickness of the final coating is about 50 mu m;
(5) Immersing the prepared probe in the step (4) into a NE solution to spontaneously polymerize the NE on the surface of the probe;
(6) And taking out the probe after 24 and h, and flushing the probe with deionized water to obtain the GO-PAN@PNE solid phase microextraction probe.
The linear range of the probe in the fish labeled with tetrodotoxin was determined using the GO-PAN@PNE probe and the optimal extraction conditions determined in example 5, and the detection limit and the quantification limit were determined, wherein the concentration of the fish labeled with tetrodotoxin was set as
5, 10, 50, 100, 250, 500, 750, 1000 ng/mL, selecting a concentration range with good linearity according to the extraction result, and calculating a detection limit and a quantitative limit according to the concentration range, wherein the signal to noise ratio of the detection limit and the quantitative limit are respectively
Selected as 3 and 10, the linear range is shown in FIG. 9, the calculated linear range is 50-1000 ng/g, the detection limit is 32 ng/g, and the quantitative limit is 150 ng/g.
Example 7
The preparation method of the GO-PAN@PNE probe for solid phase microextraction comprises the following specific steps:
(1) Activating the surface of a medical stainless steel needle to prepare GO-PAN solution and NE solution;
(2) Vertically immersing the activated stainless steel needle with the length of about 1cm at one end into the GO-PAN solution, and slowly taking out to finish the immersion;
(3) Placing the impregnated probe into an oven and heating at 90 ℃ for 3 min until DMF volatilizes, so that the coating is fixed;
(4) Repeating the immersing-heating steps in the steps (2) and (3) for a plurality of times, so that the coating is uniformly wrapped on the medical stainless steel needle as much as possible, and the thickness of the final coating is about 50 mu m;
(5) Immersing the prepared probe in the step (4) into a NE solution to spontaneously polymerize the NE on the surface of the probe;
(6) And taking out the probe after 24 and h, and flushing the probe with deionized water to obtain the GO-PAN@PNE solid phase microextraction probe.
The in-vivo detection of the tetrodotoxin in the tetrodotoxin body is carried out by using a GO-PAN@PNE probe, and as shown in figure 10, the detection steps are as follows:
(1) Anaesthetizing live globefish with eugenol;
(2) A small hole of about 1.5. 1.5 cm depth is punched on the side of the back muscle near the fin by a medical injector needle;
(3) Pulling the injector out of the small hole, and then inserting the probe into the small hole for extraction;
(4) Fishing out the fish from the water again, and taking out the probe after anesthesia again;
(5) Flushing the probe with deionized water, and desorbing the extracted tetrodotoxin with a desorption solvent;
(6) And (3) carrying out LC-MS/MS analysis on the desorption liquid to obtain the toxin content of the fugu fish in the fugu fish body.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and alterations can be made without departing from the spirit and principles of the present invention,
All simplified and equivalent substitutions are included in the scope of the present invention.
Claims (8)
1. The functionalized solid-phase microextraction probe is characterized in that the probe is obtained by coating graphene oxide-polyacrylonitrile solution on an activated stainless steel needle and spontaneously polymerizing norepinephrine solution on the surface of the probe to form PNE coating, wherein:
the graphene oxide-polyacrylonitrile solution is prepared by dissolving graphene oxide in polyacrylonitrile glue, and comprises the following components: mixing polyacrylonitrile solid and N, N-dimethylformamide in a mass ratio of 1:7-8, fully stirring to uniformly disperse the polyacrylonitrile and properly dissolve the polyacrylonitrile to form a viscous suspension, heating the suspension at 80-100 ℃ for 50-70 min to completely dissolve the polyacrylonitrile to form yellow clear transparent polyacrylonitrile gel, and cooling the mixture to room temperature; adding graphene oxide powder into N, N-dimethylformamide, and uniformly dispersing the graphene oxide powder in an ultrasonic manner, wherein the mass volume ratio of the graphene oxide powder to the N, N-dimethylformamide is 15-25mg:200-300 mu L of the graphene oxide solution is added into the polyacrylonitrile glue and stirred uniformly to obtain the graphene oxide solution;
the norepinephrine solution is dissolved in a v/v ratio of 1 by norepinephrine: 1 in a mixed solution of 10 mM of a Tri buffer solution with pH of 8.5 and methanol, and dissolving the mixture by continuous stirring;
the activated stainless steel needle is obtained by cutting a medical stainless steel needle with the diameter of 0.4-0.6 mm into small sections with the length of 2-3 cm, sequentially carrying out ultrasonic pretreatment in concentrated hydrochloric acid for 8-12 min, taking out and carrying out surface activation treatment of deionized water rinsing;
the preparation method of the functionalized solid-phase microextraction probe comprises the following steps: and (3) vertically immersing one end of the activated stainless steel needle into the graphene oxide-polyacrylonitrile solution, slowly taking out, heating at 80-100 ℃ for 2-4min until N, N-dimethylformamide is fully volatilized to fix the coating, repeating the immersing and heating steps for a plurality of times to uniformly wrap the coating on the activated stainless steel needle, immersing the activated stainless steel needle into a norepinephrine solution with the concentration of 0.2mg/mL for spontaneous polymerization 24 h, taking out, and washing with deionized water to obtain the graphene oxide-polyacrylonitrile composite material.
2. The functionalized solid phase microextraction probe according to claim 1, wherein the surface coating thickness of the functionalized solid phase microextraction probe is 50-70 μm.
3. Use of the functionalized solid phase microextraction probe according to claim 1 or 2 for the in vivo detection of tetrodotoxin, the method for in vivo detection of tetrodotoxin by the functionalized solid phase microextraction probe comprising the steps of:
(1) Anaesthetizing live globefish with eugenol;
(2) A small hole with a depth of 1.5 cm is pricked on the side surface of the back muscle near the fin by a medical injector needle;
(3) Extracting the medical injector needle from the small hole, and inserting the functional solid phase microextraction probe into the small hole for living body extraction;
(4) Fishing out the puffer fish from water, and taking out the functionalized solid-phase microextraction probe after re-anesthesia;
(5) Washing the functionalized solid phase microextraction probe with deionized water, and then desorbing the extracted tetrodotoxin with a desorption solvent;
(6) Carrying out LC-MS/MS analysis on the desorption liquid obtained in the step (5) to obtain the toxin content of the fugu fish in the fish body;
the method for anaesthetizing the puffer fish in the steps (1) and (4) comprises the following steps: adding 5 mL eugenol liquid into 5L tap water to prepare 0.1% eugenol water solution, taking out the puffer fish from water, immersing the puffer fish in the solution, and taking out the fish immediately after the body of the puffer fish is out of balance after a few seconds to finish anesthesia.
4. The use according to claim 3, wherein the detection limit of the functionalized solid phase microextraction probe on the tetrodotoxin in the tetrodotoxin muscle is 32 ng/g, the quantitative limit is 150 ng/g and the linear range is 50-1000 ng/g.
5. The use according to claim 3, wherein the in vivo extraction time is 30-70 min.
6. Use according to claim 3, wherein the desorption solvent is selected from methanol, 50% methanol, acetonitrile and 50% acetonitrile, the desorption time being 20-40 min.
7. The use according to claim 3, wherein the LC-MS/MS analysis conditions are: using an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer, wherein a mass spectrometry ion source is an electrospray ion source, separating by using an HILIC chromatographic column of Agilent technologies Co., ltd., USA, wherein a mobile phase A is an aqueous solution containing 0.1% formic acid, a mobile phase B is 0.1% acetonitrile, a flow rate is 0.4mL/min, and a column temperature is 40 ℃;
the gradient elution procedure was as follows: starting from 5% of mobile phase A and 95% of mobile phase B, after holding for 0.5min, the mobile phase A rises to 60% in 2.5min, then rises to 95% in 1min, then remains unchanged for 2min, then falls back to 5% in 0.1min, and remains unchanged for 1.9min, and the analysis time of each sample is 8min;
the mass spectrometry conditions were as follows: the sample analysis was performed using electrospray ion source positive ion mode, gas curtain gas of 35psi, ionization voltage of 5500V, desolvation temperature of 500 ℃, atomizing gas of 55psi, auxiliary heating gas of 55psi, multiple reaction monitoring.
8. The use according to claim 3, wherein in the step (6), the method for quantifying the concentration of the tetrodotoxin in the desorption solution by using the external standard method comprises the following steps: dissolving tetrodotoxin in a small amount of acetic acid aqueous solution with v/v of 1%, preparing tetrodotoxin standard gradient solutions with concentrations of 0.1, 0.5, 1, 5, 10, 50 and 100 ng/mL respectively by using methanol to fix the volume, analyzing the tetrodotoxin standard gradient solution by using LC-MS/MS, drawing a standard curve of the tetrodotoxin by using the concentration as an x axis and the peak area as a y axis, and quantifying the tetrodotoxin concentration of the desorption liquid.
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