CN107014888A - The method that trace materials in sample to be tested is analyzed using molecular engram film electrospray ionization mass spectrometry - Google Patents
The method that trace materials in sample to be tested is analyzed using molecular engram film electrospray ionization mass spectrometry Download PDFInfo
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- CN107014888A CN107014888A CN201710212298.6A CN201710212298A CN107014888A CN 107014888 A CN107014888 A CN 107014888A CN 201710212298 A CN201710212298 A CN 201710212298A CN 107014888 A CN107014888 A CN 107014888A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
Abstract
The method that the present invention provides trace materials in a kind of utilization molecular engram film electrospray ionization mass spectrometry analysis sample to be tested, including：1) microfiltration membranes surface modification and activation；2) it will modify and immersed with the microfiltration membranes after activation in the reaction dissolvent containing template molecule, function monomer, crosslinking agent and initiator, synthetic molecules blotting membrane is reacted in deoxidation；3) by step 2) synthesis molecular engram film immersion eluting solvent in, removing template molecule is rocked at room temperature；4) by step 3) molecular engram film add sample to be tested in, 10 30min are rocked at room temperature, take out, molecular engram film is detected using molecular engram film electron spray ionisation device.The present invention realizes real-time analysis of the actual sample under high sensitivity, has high application value in food security, blood concentration, antibiotic residue and residues of pesticides field, and have broad application prospects without Sample pretreatment.
Description
Technical field
The present invention relates to molecular engram film electrospray ionization mass spectrometry (Molecularly imprinted membrane
Electrospray ionization mass spectrometry, MIM-ESI-MS) exploitation and application, specifically, relate to
And the workflow of molecular engram film electrospray ionization mass spectrometry, and application of the technology in multiple fields, such as in food
Safety, blood concentration, the quick detection of the actual sample to be tested of antibiotic residue and residues of pesticides.
Background technology
Mass spectrum (Mass spectrometry, MS) is a kind of powerful analysis tool, with high sensitivity, high-resolution,
The advantage such as sample analysis consumption is small, is widely used in different fields.In recent years, open type ionization technique (ambient
Ionization, AM) turn into study hotspot and continue to develop, the technology shortens or avoided the pre-treatment of complex samples
Journey, makes it possible to apply mass-spectrometric technique quick detection complex samples.Common open type ionization technique includes：Parse electron spray
Ionize (DESI), Direct Analysis in Real Time mass spectrum (DART), paper spraying ionization (PS) and film electron spray ionisation (MESI) etc..
MESI is the new open type Ionization Techniques In Mass Spectrometry that applicant had previously developed.The technology is by biomembrane combination mass spectrum
Ion gun, three-dimensional separation in real time can be realized during sample analysis, matrix interference is effectively eliminated, more conventional method is very big
Ground improves detection sensitivity.Although film electron spray ionisation technology has an above-mentioned advantage, but the trace in analysis complex samples
During test substance, expect to obtain lower detection limit and broader dynamic range is still challenging.
In order to further lift MESI performance, plan it and be combined with separation and concentration technology, develop new open wide
Formula ionization techniques.Method for separating and concentrating common at present is varied, such as SPME, molecular engram, and its application is all
By wide coverage.Wherein, molecular engram be using molecularly imprinted polymer (Molecular imprinting polymer,
MIP), the strategy of specific enrichment is carried out to test substance.MIP is that one kind passes through covalent bond using template molecule and function monomer
Or non-covalent bond combines the polymer formed, elutes after template molecule, leaves to certain specific template molecule and its class
There is the hole of selection identity like thing.Wherein, pseudo- template molecule refers to there is similar identification or trace with template molecule
Site, similar molecular size and shape, elution are removed after pseudo- template molecule, and the hole of formation still has selection identity.
The content of the invention
The fast qualitative quantitative analysis on the premise of high sensitivity is ensured can not be met in order to solve open type ionization technique
The problem of, the present invention provides a kind of with high sensitivity and without the new open type ionization technique of Sample pretreatment, molecule print
Mark film electron spray ionisation technology (MIM-ESI), applied to food security, blood concentration, antibiotic residue and residues of pesticides.
Present invention firstly provides a kind of microfiltration membranes surface modification and the method for activation, comprise the following steps：
1) microfiltration membranes are immersed in aqueous slkali, after 55-65 DEG C of processing, film surface is cleaned with pure water；
2) again by step 1) in the aqueous solution of the obtained film immersion containing acrylic acid and potassium peroxydisulfate, after deoxidation, 65-75 DEG C
Lower isothermal reaction；
3) finally by step 2) obtained microfiltration membranes immerse pure water, acetonitrile, 0.15molL successively-1AIBN acetonitrile solutions
In, volatilize after solvent and to immediately enter film in molecular engram reaction solution.
Microfiltration membranes of the present invention include but is not limited to pvdf membrane, PTFE film or cellulose membrane.
The present invention microfiltration membranes surface modification with activation method in, step 1) in aqueous slkali be the 3%NaOH aqueous solution；
55-65 DEG C of processing time is 10-15h, it is preferable that 60 DEG C of processing 12h.
The microfiltration membranes surface modification of the present invention with the method for activation, step 2) in film is entered containing 10% acrylic acid and
In the aqueous solution of 1% potassium peroxydisulfate, deoxidation；Isothermal reaction 4-6h under 65-75 DEG C of nitrogen environment, it is preferable that 70 DEG C of nitrogen environments
Lower isothermal reaction 5h.
The present invention provides trace materials in a kind of utilization molecular engram film electrospray ionization mass spectrometry analysis sample to be tested
Method, comprises the following steps：
1) surface modification and the activation of microfiltration membranes are carried out according to the method described above；
2) synthetic molecules blotting membrane：Surface modification is contained into template molecule, function list with the microfiltration membranes immersion after activation
In the molecular engram reaction solution of body, crosslinking agent and initiator, deoxidation, constant temperature polymerize under nitrogen environment；The template molecule according to
Testing molecule selects similar substance；The reaction dissolvent is chloroform and/or methanol；
3) eluted template molecule：By step 2) in obtained molecular engram film immersion eluting solvent to remove template point
Son；
4) molecular engram film for removing template molecule is immersed in sample to be tested, taken out, using molecular engram film electron spray
Ionization device is detected to molecular engram film.
In one embodiment of the invention, the above method is used to detect the antibiotic residue in sample to be tested.
The utilization molecular engram film electrospray ionization mass spectrometry that the present invention is provided directly quickly is analyzed anti-in sample to be tested
The step 2 of the method for raw element residual) in, template molecule is Norfloxacin (NFLX), and function monomer is MAA, and crosslinking agent is
EGDMA, initiator is AIBN, and NFLX and MAA, EGDMA ratio are 1:4:10.Step 2) in, reaction solution is chloroform：Methanol
(v:V=5:1), liquor capacity is advisable with can fully dissolve preceding four kinds of materials (NFLX, MAA, EGDMA, AIBN).
Further, step 2) in, concrete operations are：First, template molecule is fully dissolved with reaction dissolvent, then added
Function monomer, at room temperature prepolymerization 1h；Crosslinking agent and initiator are added, the PVDF of surface modification and activation is added after dissolving
1-3h is reacted in film, deoxidation at room temperature.
Further, step 2) react at room temperature after 1-3h, film is moved on into 60 DEG C of reactions of constant temperature under nitrogen environment
24h；After reaction terminates, according to step 3) elution removes template molecule, then with pure water to neutrality, then eluted simultaneously with acetone
Volatilize, be stored in standby in drier.
Step 3) concrete operations be：0.5-1h is rocked, solvent is changed and is repeated several times, remove template molecule.The elution
Solvent is acetic acid and methanol；Elution time 0.5-1h, changes solvent and is repeated several times, it is preferable that 30min is eluted at room temperature, altogether weight
It is multiple 4 times.
Step 4) sample to be tested is liquid, or when sample to be tested is solid, is dissolved, made with suitable solvent
Liquid.Inventor's discovery, step 4), i.e. in the liquid system that molecular engram film is added to sample to be tested, Percentage bound-time is big
Cause situation is 5-15min linear rises, and 15-30min rises slow, the close balances of 30-60min.It is therefore preferred that step 4)
Absorption rocks the time for 10-30min, it is highly preferred that the time is 20min.
Present invention also offers application of the above method in field of food safety, the lean meat in biological specimen is simulated in detection
Refined salt Clenbuterol, special instruction, according to different testing molecules, the template molecule of corresponding molecular engram film is selected not
Together, it is different from the mol ratio of function monomer and crosslinking agent and different from reaction dissolvent.
Present invention also offers application of the above method in blood concentration detection, the first ammonia in biological specimen is simulated in detection
Pterin, special instruction, according to different testing molecules, the template molecule selection of corresponding molecular engram film is different, with function list
Body is different with the mol ratio of crosslinking agent, and different from reaction dissolvent.
Present invention also offers the above method in the detection of soil sample Pesticide Residues, detect in simulation biological specimen
Chlopyrifos, special instruction, according to different testing molecules, the template molecule selection of corresponding molecular engram film is different, with function
Monomer is different with the mol ratio of crosslinking agent, and different from reaction dissolvent.
The quick of food security, blood concentration, antibiotic residue and residues of pesticides is may be implemented in using the method for the present invention
Real-time qualitative, quantitative analysis.In addition to aforementioned four application, method of the invention can design relative according to the property of test substance
The molecular engram film answered, to meet analysis, therefore the inventive method has broad prospect of application.
There is provided utilize molecular engram film electrospray ionization mass spectrometry in food security in an embodiment of the present invention：It is thin
Meat refined salt Clenbuterol；Blood concentration：Methotrexate (MTX)；Antibiotic residue：Ciprofloxacin and residues of pesticides：Chlopyrifos is determined
Property, quantitative detecting method, it comprises the following steps：
1) molecule of the synthesis using Ractopamine, methoxybenzyl aminopyrimidine, Norfloxacin and tolelofos-methyl as templated synthesis
Blotting membrane, and elute the template molecule removed on molecular engram film；
2) molecular engram film and testing molecule are combined；
3) using molecular engram film electrospray ionization mass spectrometry (MIM-ESI-MS) to combining to be measured point on molecular engram film
Son, clenobuterol hydrochloride, methotrexate (MTX), Ciprofloxacin and chlopyrifos are detected.
Wherein, in order to ensure the specific adsorption and enrichment function of molecular engram film, it is necessary to take suitable method to synthesize
Corresponding molecular engram film.
Step 1) in prepare the method for molecular engram film, be specially：
Template molecule Ractopamine (Rac), in function monomer MAA, crosslinking agent EGDMA and initiator A IBN participation
Under, add the microfiltration membranes of surface modification and activation.Finally, reacted molecular engram film is obtained.It is molten with 10% acetic acid methanol
Liquid cyclic washing is to remove template molecule, and washing is volatilized, drying for standby.
Template molecule TMP (TMP), in function monomer MAA, crosslinking agent EGDMA and initiator A IBN presence,
Add the microfiltration membranes of surface modification and activation.Finally, reacted molecular engram film is obtained.Washed with 20% acetic acid methanol solution
Template molecule is washed away, washing is volatilized, drying for standby.
Template molecule Norfloxacin (NFLX), in function monomer MAA, crosslinking agent EGDMA and initiator A IBN presence,
Add the microfiltration membranes of surface modification and activation.Finally, reacted molecular engram film is obtained.Washed with 20% acetic acid methanol solution
Template molecule is washed away, washing is volatilized, drying for standby.
Template molecule tolelofos-methyl (TCFM), in function monomer MAA, crosslinking agent EGDMA and initiator A IBN participation
Under, add the microfiltration membranes of surface modification and activation.Finally, reacted molecular engram film is obtained.It is molten with 20% acetic acid methanol
Liquid washing removes template molecule, and washing is volatilized, drying for standby.
Step 2) described in the combination of molecular engram film and testing molecule carried out in analog sample system.With reference to mode
For：By test substance, first dissolving is made into mother liquor in methanol or acetonitrile solution, is then added to analog sample according to experiment demand
In system, molecular engram film is eventually adding, 20min is rocked at room temperature and is combined.
Step 3) in using molecular engram film electron spray mass spectrometry (MIM-ESI-MS) detected that equipment therefor is molecule
Blotting membrane electric spray ion source-mass spectrograph, eluting solvent used is to be added on 0.1% formic acid methanol solution, film after eluting solvent,
Through electron spray ionisation device make sample elution dissolve to be formed spray and ionize, detected subsequently into mass spectrometer inlet.
Film electrospray ion source device of the present invention can be (entitled using Application No. 201410528504.0
Mass spectrograph for the film electrospray ion source device of mass spectral analysis, ioning method and comprising the ion source device) invention
The film electrospray ion source device referred in application.Ion source device includes film and conductive component.
Molecular engram film electro-spray ionization method of the present invention for mass spectral analysis, including：By molecular engram film
Tip point to mass spectrograph injection port；High-tension electricity is applied to the molecular engram film by a conductive component；Printed to the molecule
Eluting solvent is added on mark film；In the presence of high voltage and the eluting solvent, the testing sample formation spraying and ion
Change.
Apply 1-5kV high-tension electricities, preferably 2.5kV to the molecular engram film.
The rinse solvent is the mixture of methanol and formic acid.The volume ratio of wherein described methanol and formic acid is 100：0.1.
Second order mses carry out structure elucidation using collision induced dissociation (CID) to testing molecule.The quality of parent ion sampling
Scope chooses 2.0m/z, and collision energy chooses 25%.
Present invention also offers a kind of molecular engram film detected suitable for molecular engram film electrospray ionization mass spectrometry, lead to
Following methods are crossed to prepare：Surface modification is added with the microfiltration membranes after activation and contains template molecule, function monomer, crosslinking
Handled in agent, the reaction dissolvent of initiator, nitrogen deoxidation；The selection principle of the template molecule be and testing molecule analog
Matter；The reaction dissolvent be chloroform and/or methanol described in function monomer be MAA, crosslinking agent is EGDMA, and initiator is AIBN；
Reaction dissolvent is chloroform and/or methanol；
Then concrete operations add function monomer, prepolymerization 1h fully to dissolve template molecule with reaction dissolvent；Then plus
Enter crosslinking agent and initiator, the microfiltration membranes of surface modification and activation are added after dissolving, 1-3h is reacted in deoxidation at room temperature, will
Reacted microfiltration membranes are transferred to 50-70 DEG C of reaction 24-30h of constant temperature under nitrogen environment；After reaction terminates, 10%-20% second is used
Sour methanol solution cyclic washing, then with pure water to neutrality, washed and volatilized with acetone, it is standby.
The present invention realizes the hydrochloric acid gram human relations to being added in simulation biological specimen using molecular engram film electrospray ionization mass spectrometry
Special sieve, methotrexate (MTX), Ciprofloxacin and the qualitative and quantitative analysis of chlopyrifos.The present invention adsorbs complicated sample using molecular engram film
Low concentration test substance in this, greatly improves detection sensitivity, and experiment is time-consuming less than 30min.Generally realize high
To the quick detection of test substance under sensitivity.
The present invention combines molecularly imprinted polymer and film electron spray ionisation technology, develops new mass ions skill
Art-molecular engram film electron spray ionisation (MIM-ESI).The molecularly imprinted polymer on commercialization microfiltration membranes surface bond, is formed
Molecular engram film.Ionization and mass spectral analysis in real time are used directly for after film absorption sample.Due to the spy of molecular engram film
Opposite sex identification and enrichment, MIM-ESI-MS can realize the real-time mass spectral analysis of trace materials in complex samples, with
NanoESI or the contrast of other open type ionization techniques, sensitivity can improve 10-50 times.The inventive method can also be according to be measured
Structure and property synthesize corresponding molecular engram film, it is adaptable to different field, have broad application prospects.
The present invention is realized residual in food security, blood concentration, antibiotic using molecular engram film electrospray ionization mass spectrometry
The detection and analysis with residues of pesticides field is stayed, the technology can complete the real-time Mass Spectrometer Method in complex samples, without sample
This pre-treatment, mass analysis time is less than 1min, and integral experiment is time-consuming to be less than 30min.And the present invention can also be by changing
For the molecular engram film of different test substances so as to realize the detection of different material, application prospect is extensive, except in the present invention
Refer to four fields outside, can also expand applied to clinic, criminal investigation, the actual sample detection in the field such as energy, therefore molecule
Blotting membrane electrospray ionization mass spectrometry has a good application prospect.
Brief description of the drawings
Figure 1A-Fig. 1 F are the scanning electron microscope (SEM) photograph and infrared spectrogram of molecular engram film of the present invention, and Figure 1A-Fig. 1 F are respectively sky
The scanning electron microscope (SEM) photograph of white pvdf membrane；With reference to the molecular engram film of template molecule Ractopamine；Elution removes template molecule Rec
Molecular engram film after dopamine；The infrared spectrogram of blank pvdf membrane；With reference to the molecular engram of template molecule Ractopamine
The infrared spectrogram of film；Elution removes the infrared spectrogram of the molecular engram film of template molecule Ractopamine.
Fig. 2 is the structural representation that molecular engram film electrospray ion source device of the present invention is operated.1 represents molecular engram
Film, 2 represent conductive component, are commonly used for conductive copper folder.
Fig. 3 is the result using the inventive method MIM-ESI-MS analysis clenbuterol hydrochloride clenobuterol hydrochlorides (CLE), wherein, A
Figure：CLE standard curve in urine specimen；B schemes：CLE (0.1ngmL in urine specimen-1) first mass spectrometric figure；C schemes：Urine
CLE (0.1ngmL in sample-1) second order mses figure；D schemes：The second order mses figure of dummy.
Fig. 4 is (to be poisoned with poison using the inventive method MIM-ESI-MS analysis blood concentrations (methotrexate (MTX), MTX), residues of pesticides
Tick, CPF) and antibiotic residue (Ciprofloxacin, CPFX), wherein, MTX standard curve in A figures and D figures respectively blood sample
With second order mses figure；B schemes and E figures are respectively the standard curve and second order mses figure of CPF in soil sample；C schemes and F figures are respectively
The standard curve and second order mses figure of CPFX in milk sample.
Fig. 5 is the specific adsorption contrast of molecular engram film and non-imprinted membrane.
Fig. 6 A- Fig. 6 F are three kinds of ion gun method comparative analysis figures of Ciprofloxacin in simulation biological specimen, Fig. 6 A- Fig. 6 B
Respectively：Utilize the standard curve of Ciprofloxacin in tri- kinds of ion gun analysis milk of nanoESI, PS and MIM-ESI；Utilize MIM-
The firsts and seconds mass spectrogram of Ciprofloxacin in ESI analysis milk；Fig. 6 C and Fig. 6 D are to utilize nanoESI analysis milk middle ring third
Sha Xing firsts and seconds mass spectrogram；Fig. 6 E and Fig. 6 F are the firsts and seconds mass spectrum that Ciprofloxacin in milk is analyzed using PS
Figure.
Embodiment
Below with reference to the accompanying drawings embodiments of the invention are illustrated.Retouched in a kind of accompanying drawing or embodiment of the present invention
The element and feature that the element and feature stated can be shown in one or more other accompanying drawings or embodiment are combined.Should
Work as attention, for purposes of clarity, eliminated in accompanying drawing and explanation known to unrelated to the invention, those of ordinary skill in the art
Part or processing expression and description.The present invention is described further below in conjunction with the accompanying drawings.
The principle of molecular imprinting technology, washes away and is left after template molecule to certain specific template molecule and the like tool
There is the hole of selection identity.The remaining influence not eluted completely in order to avoid template molecule quantifying subsequently, is employed herein
The pattern of template molecule and the non-same molecule of testing molecule.Also, some template molecules, such as methotrexate (MTX), in conventional molecule
Poor solubility in trace reaction dissolvent and be difficult to synthesize, synthetic molecules blotting membrane is used for using pseudo- template molecule here.
Material therefor and reagent in the present invention.
The reagent list of table 1
The molecular engram film surface modification of embodiment 1 is with activation (by taking pvdf membrane as an example)
In the NaOH aqueous solution that pvdf membrane is immersed to 3%, 12h is handled at 60 DEG C, film surface is cleaned with pure water, then will
In the treated aqueous solution of the film immersion containing 10% acrylic acid and 1% potassium peroxydisulfate, after nitrogen deoxidation, the constant temperature at 70 DEG C
React 5h.Then, the pvdf membrane handled well is immersed in 30min, 0.15molL in 1h in pure water, acetonitrile successively-1AIBN acetonitriles
20min is activated in solution, is finally volatilized acetonitrile and is immersed immediately in molecular engram reaction solution.
The synthesis of the Ractopamine molecular engram film of embodiment 2
0.0052g template molecules Ractopamine (Rac) is weighed, 10.43 μ L function monomer MAA is added, uses 0.06mL first
After alcohol fully dissolves, 0.3mL chloroforms are added, prepolymerization 1h at room temperature after sealing.Then 0.1178mL crosslinking agents EGDMA is added
With 0.0060g initiator A IBN, until completely dissolved, surface modification made from embodiment 1 and the pvdf membrane of activation are added, is surpassed
Sound 10min removes oxygen, and 2h is reacted at room temperature.Finally, reacted pvdf membrane is transferred to 65 DEG C of constant temperature under nitrogen environment
React 24h.After reaction terminates, with 10% acetic acid methanol solution cyclic washing to remove template molecule, then with pure water extremely
Neutrality, is washed and volatilized with acetone, molecular engram film is stored in standby in drier.
The synthesis of the TMP molecular engram film of embodiment 3
0.0232g template molecules TMP (TMP) is weighed, 34.2 μ L function monomer MAA is added, is filled with 1.5mL chloroforms
After point dissolving, prepolymerization 1h at room temperature after sealing.Then 0.4526mL crosslinking agent EGDMA and 0.0050g initiators are added
AIBN, until completely dissolved, adds surface modification made from embodiment 1 and the pvdf membrane of activation, ultrasonic 10min is except deoxidation
Gas, reacts 2h at room temperature.Finally, reacted pvdf membrane is transferred to 55 DEG C of reaction 24h of constant temperature under nitrogen environment.Reaction knot
Shu Hou, with 20% acetic acid methanol solution cyclic washing to remove template molecule, then with pure water to neutrality, is washed with acetone
And volatilize, molecular engram film is stored in standby in drier.
The synthesis of the norfloxacin molecular imprinted film of embodiment 4
0.0319g template molecules Norfloxacin (NFLX) is weighed, 34.2 μ L function monomer MAA is added, uses 1.0mL chloroforms:
Methanol (4:1) after mixed solution fully dissolves, prepolymerization 1h at room temperature after sealing.Then 0.1886mL crosslinking agents are added
EGDMA and 0.0050g initiator A IBN, until completely dissolved, add surface modification made from embodiment 1 and the PVDF of activation
Film, ultrasonic 10min removes oxygen, and 2h is reacted at room temperature.Finally, reacted pvdf membrane is transferred to constant temperature under nitrogen environment
60 DEG C of reaction 24h.After reaction terminates, with 20% acetic acid methanol solution cyclic washing to remove template molecule, then washed with pure water
Wash to neutrality, washed and volatilized with acetone, molecular engram film is stored in standby in drier.
The synthesis of the tolelofos-methyl molecular engram film of embodiment 5
0.04517g template molecules tolelofos-methyl (TCFM) is weighed, 51.2 μ L function monomer MAA is added, uses 1.5mL second
Nitrile fully dissolves, prepolymerization 10h at 4 DEG C after sealing.Then 565.8 μ L crosslinking agent EGDMA and 0.0030g initiators are added
AIBN, until completely dissolved, adds surface modification made from embodiment 1 and the pvdf membrane of activation, ultrasonic 10min is except deoxidation
Gas, reacts 2h at room temperature.Finally, reacted pvdf membrane is transferred to 60 DEG C of reaction 24h of constant temperature under nitrogen environment.Reaction knot
Shu Hou, with 20% acetic acid methanol solution cyclic washing to remove template molecule, then with pure water to neutrality, is washed with acetone
And volatilize, molecular engram film is stored in standby in drier.
The synthesis of the non-imprinted membrane of embodiment 6
In addition to template molecule is added without, remaining step is identical with above-mentioned synthetic molecules blot procedure.
Embodiment 7 utilizes the hydrochloric acid in the directly quick analysis mode biological specimen of molecular engram film electrospray ionization mass spectrometry
Clenbuterol, methotrexate (MTX), Ciprofloxacin and the method for chlopyrifos are set up
1st, molecular engram film configuration of surface and property representation
By ESEM (Hitachi SU8010, Japan) and infrared spectrum (Tensor II, German Bruker) to synthesis
Four kinds of molecular engram film configurations of surface and property representation.
So that Ractopamine is the molecular engram film that template molecule is synthesized as an example, the scanning electron microscope (SEM) photograph of molecular engram film is shown
Show, relative to blank pvdf membrane (Figure 1A), molecular engram film (Figure 1B) surface can see apparent polymer network structure, say
Bright molecular engram material is bonded in pvdf membrane surface.The molecular engram film (Fig. 1 C) removed after template molecule is eluted simultaneously, can be with
See thin PVDF fibers.Infrared spectrogram to molecular engram film is shown, relative to blank pvdf membrane (Fig. 1 D), combines Lay
The molecular engram film (Fig. 1 E) of gram dopamine.It is negative with higher electricity because the N in Rac N-H is connected in ionic bond form with H
Property, it is strong proton donor, can acts on forming stable hydrogen bond with the COOH in MAA.After both results, infrared spectrum
Changed, vibration peak is moved to high wave number, blue shift occurs, so 1698cm-1For N-H deformation vibration the absworption peak,
2945cm-1For-OH characteristic absorption peak, 799,753cm-1, to sum up, can be with from infrared spectrum for characteristic absorption peak on phenyl ring
Find out, stable hydrogen bond is formd between Ractopamine and MAA, illustrates the molecular engram material key using Rac as template molecule
Close on pvdf membrane.Fig. 1 F remove the infrared spectrum of molecular engram film after Rac for elution, it can be seen that Rac is removed substantially, can
For subsequent adsorbtion experiment.
2nd, molecular engram film and testing molecule are combined in simulation biological specimen
Test substance is dissolved in methanol or acetonitrile and is made into mother liquor.
Clenbuterol hydrochloride can be detected in the urine using the pig of clenbuterol hydrochloride, human urine is chosen here biological as simulation
Sample, the biotic environment of simulation pig urine.Methylpterin is widely used as cancer therapy drug in chemotherapy, is present in the blood of patient
In, the blood of biological rat is chosen here as simulation biological specimen, simulates the biotic environment of patient blood.Ciprofloxacin conduct
Antibiotic medicine is widely used in food and medicine, and milk is chosen here as simulation biological specimen.Chlopyrifos is used as organophosphor
Agricultural chemicals is used in agricultural production, and remains in soil or crops, and soil is chosen here as simulation biological specimen.
0.1-100000ngmL is made into 1.5mL simulation biological specimens-1Concentration gradient (clenobuterol hydrochloride), 0.5-10000ng
mL-1Concentration gradient (methotrexate (MTX)), 1-1000ngmL-1Concentration gradient (chlopyrifos), 1-5000ngmL-1Concentration gradient (ring
Third husky star).It is corresponding respectively to add the obtained molecular engram films (fan-shaped radius about 5mm, 45 ° of central angle) of fan-shaped embodiment 2-5,
Absorption 20min is rocked at room temperature.
3rd, the molecular engram film electrospray ionization mass spectrometry (MIM-ESI-MS) of simulation biological specimen is quickly analyzed
All MIM-ESI-MS experiments pass through Bruker HCT mass spectrographs (German Bruker Daltonics Co., Ltds)
Carry out.
Used molecular engram film electron spray ionisation device (MIM-ESI-MS), as shown in Fig. 2 including：Molecular engram film
1；Conductive component 2, molecular engram film 1 is connected after being combined described in embodiment 7.2 with testing molecule with conductive component 2, conductive
Part 2 is good conductive body, conventional for copper clip.
Molecular engram film 1 has at least one tip, i.e. sample introduction end, by the tip just to mass spectrograph injection port, and make conduction
Part 2, tip, mass spectrograph injection port are located along the same line, and are easy to realize the electron spray of sample in mass spectrograph injection port.
The tip of molecular engram film 1 can be an acute angle, right angle or obtuse angle, and its angle is preferably 40-150 °, to dialysis membrane
2 concrete shape is not limited, and it can have a tip to be anterior, and rear portion is in the irregular shape of circular arc；Can also be
The triangle of rule, preferably 45 ° sectors, radius can be 5-10mm, preferably 5-7mm.
Nitrogen is used for dry gas (flow velocity 10Lmin-1；150 DEG C of temperature).Positive ion mode, capillary voltage for-
2.5kV.As shown in Fig. 2 molecular engram film electrospray ionization mass spectrometry device (MIM-ESI-MS) is treated will to have been adsorbed with metal clip
The molecular engram film for surveying material is fixed on mass spectrum injection port front end, the tip alignment mass spectrum entrance of triangle film, and keeps therewith
About 5mm distance.And plus 2.5kV DC voltage, 8 microlitres of eluting solvent is added on film (methanol：Formic acid volume ratio is
100：0.1).In the presence of high voltage electric field and eluting solvent, nose motion from solution to be measured to molecular engram film, in front end
One jiao produces sample ions and forms spraying.
Human urine is chosen as simulation biological specimen, the biotic environment of simulation pig urine.Salt is added in 1.5mL urine specimens
Clenbuterol mother liquor, is made into 0.1-100000ngmL-1Concentration gradient, by embodiment it is 2-in-1 into molecular engram film soak completely
Enter in solution, 20min is rocked at room temperature.Analyzed followed by MIM-ESI-MS, as a result as shown in Figure 3.Fig. 3 A map analysis urine
Clenobuterol hydrochloride standard curve in liquid, the range of linearity is 0.1-100000ngmL-1, coefficient correlation (r2) it is 0.9973, LOD
It is respectively 0.02ngmL with LOQ-1And 0.1ngmL-1.Clenobuterol hydrochloride concentration is in Fig. 3 B-C diagram urines
0.1ng·mL-1Firsts and seconds mass spectrogram, successfully be detected clenobuterol hydrochloride mother and sons' ion pair (m/z 277 → 259,
203).Fig. 3 D, which is shown in negative sample, does not detect clenobuterol hydrochloride.
In addition to the application of field of food safety, MIM-ESI-MS is also applied in other field, such as blood concentration, anti-
Raw element residual and residues of pesticides.As shown in figure 4, using in CPF and milk in MTX, soil in MIM-ESI-MS analysis blood
CPFX result.Shown in Fig. 4 A-C, the standard curve in analysis blood in MTX, soil in CPF and milk obtained by CPFX passes through
Molecular engram film is to the enrichment of testing molecule, and above MTX, CPF and CPFX detection limit is respectively 0.5ngmL-1,
1.0ng·mL-1And 1.0ngmL-1.Fig. 4 D-4F show MTX, CPF and CPFX second order mses figure, successfully be detected blood sample
In MTX (mother and sons' ion pair m/z 455 → 308), CPF (mother and sons' ion pair m/z 350 → 335) and milk in soil sample
CPFX (mother and sons' ion pair m/z 332 → 313,287) in sample.
2nd, molecular engram film specific recognition absorption checking
Investigate specific adsorption effect of the molecular engram film to testing molecule of synthesis.With in analysis mode biological specimen
Exemplified by Ciprofloxacin, Ciprofloxacin mother liquor, three concentration level (high concentrations are added in milk analog sample：1μg·mL-1；In
Concentration：10ng·mL-1；Low concentration：1ng·mL-1).The molecular engram film that embodiment 4 and embodiment 6 are synthesized immerses respectively to be added
Plus in the milk sample of Ciprofloxacin, room temperature is rocked after absorption 20min, is analyzed using MIM-ESI-MS.As shown in figure 5, molecule
The adsorption effect of blotting membrane is higher than non-imprinted membrane, and both lower differences of Ciprofloxacin Concentration are bigger, illustrate molecular engram
The specific recognition effect of film is obvious.
3rd, MIM-ESI-MS and nanoESI mass spectrographies and paper ESI-MSr (PS) comparative analysis
Test substance is dissolved in methanol or acetonitrile and is made into mother liquor, then 1- is made into 1.5mL simulation biosystems
5000ng·mL-1Concentration gradient (Ciprofloxacin).NanoESI mass spectrographies and the analysis of paper ESI-MSr is respectively adopted, and to three
The result of the method for kind is analyzed.
NanoESI mass spectrographies, use nitrogen for dry gas (flow velocity 10Lmin-1；150 DEG C of temperature).Positive ion mode,
Capillary voltage is -1.2kV.The tip alignment mass spectrum entrance of capillary, and about 5mm distance is kept therewith.
PS mass spectrographies, mass spectrum injection port front end is fixed on metal clip by triangle chromatographic paper, triangle paper it is sophisticated right
Quasi- mass spectrum entrance, and about 5mm distance is kept therewith.And plus 4.5kV DC voltage, 8 microlitres of eluting solvent is added to
(methanol on film：Formic acid volume ratio is 100：0.1).In the presence of high voltage electric field and eluting solvent, solution to be measured prints to molecule
The nose motion of mark film, one jiao in front end produces sample ions and forms spraying.
MIM-ESI-MS principle is similar to MESI and PS.By molecular engram film to the specific adsorption of testing molecule and
Enrichment, MIM-ESI quantitative detection limit is lower compared with other open type ionization techniques.With the ring in analysis mode biological specimen
Exemplified by third husky star, three kinds of analysis methods are contrasted：MIM-ESI, PS and nanoESI.Linear models of the MIM-ESI compared with other two methods
Enclose wider (Fig. 6 A).Fig. 6 B- Fig. 6 F are the firsts and seconds mass spectrogram result that Ciprofloxacin is analyzed using three kinds of methods, and ring third is husky
Mother and sons' ion pair (m/z 332 → 313,287) of star, three kinds of methods of contrast are it can be found that MIM-ESI LOQ is lower
1.0ng·mL-1, and nanoESI and PS are respectively 50ngmL-1And 10ngmL-1.Illustrate the effectively suction of molecular engram film
Attached and be enriched testing molecule, MIM-ESI-MS is adapted to the analysis of trace object in complex environment.
The Method validation of embodiment 8
The present invention investigates the mass signal obtained after the test substance solution in molecular engram film absorption simulation biological specimen
Intensity, so as to obtain the linear of standard curve, LOD and LOQ.As shown in table 2,4 testing molecules are in 0.1-100000ngmL-1
(CLE), 0.5-10000ngmL-1(MTX), 1-1000ngmL-1(CPF), 1-5000ngmL-1(CPFX) the range of linearity
Coefficient correlation (r2) it is 0.9973,0.9982,0.9931,0.9948.The LOD of clenobuterol hydrochloride in urine analog sample
For 0.02ngmL-1, LOQ is 0.1ngmL-1；The LOD of methotrexate (MTX) in blood analog sample is 0.1ngmL-1, LOQ
For 0.5ngmL-1；The LOD of chlopyrifos in Simulated Soil sample is 0.5ngmL-1, LOQ is 1.0ngmL-1；Milk mould
The LOD for intending the Ciprofloxacin in sample is 0.5ngmL-1, LOQ is 1.0ngmL-1.The present invention is to utilizing MIM-ESI-MS points
The Method validation of four kinds of test substances, including the range of linearity, LOD, LOQ, the degree of accuracy, precision and the rate of recovery are analysed, as a result such as
Shown in table 3.The MIM-ESI-MS of this method has very in the degree of accuracy in the daytime and the in a few days degree of accuracy, day to day precision and withinday precision
Good collimation, and this method can obtain the good rate of recovery, for the trace materials in analysis complex samples have compared with
Good quantitative analysis ability.
The range of linearity, minimum detectability and the minimum quantitative limit of 2 four testing molecules of table
The degree of accuracy of 3 four testing molecules of table, precision, the rate of recovery
Although the present invention and its advantage has been described in detail it should be appreciated that without departing from by appended claim
Various changes can be carried out in the case of the spirit and scope of the present invention limited, substitutes and converts.Moreover, the model of the application
Enclose the process described by specification of being not limited only to, equipment, means, the specific embodiment of method and steps.In the art is common
Technical staff be will readily appreciate that from the disclosure, and execution and corresponding reality described herein can be used according to the present invention
Apply the essentially identical function of example or obtain process essentially identical with it result, existing and that future is to be developed, equipment,
Means, method or step.Therefore, appended claim includes such process, equipment, hand in the range of being directed at them
Section, method or step.
Claims (10)
1. a kind of microfiltration membranes surface modification and the method for activation, comprise the following steps：
1) microfiltration membranes are immersed in aqueous slkali, after 55-65 DEG C of processing, film surface is cleaned with pure water；
2) film is immersed in the aqueous solution containing acrylic acid and potassium peroxydisulfate again, after deoxidation, isothermal reaction at 65-75 DEG C；
3) step 2) microfiltration membranes be immersed in pure water, acetonitrile, 0.15molL successively-1Activated in AIBN acetonitrile solutions, finally
Acetonitrile is volatilized to immerse immediately in molecular engram reaction solution.
2. the method as described in claim 1, it is characterised in that the microfiltration membranes are pvdf membrane, PTFE film or cellulose membrane；Step
It is rapid 1) in aqueous slkali be 3% the NaOH aqueous solution；Step 1) 55-65 DEG C of processing time be 10-15h.
3. the method as described in claim 1, it is characterised in that step 2) in film immersion is contained into 10% acrylic acid and 1% mistake
In the aqueous solution of potassium sulfate, after deoxidation, isothermal reaction 4-6h under 65-75 DEG C of nitrogen environment.
4. a kind of method of trace materials in utilization molecular engram film electrospray ionization mass spectrometry analysis sample to be tested, including following step
Suddenly：
1) any described methods of claim 1-3 carry out surface modification and the activation of microfiltration membranes；
2) template molecule blotting membrane is synthesized：Surface modification is contained into template molecule, function list with the microfiltration membranes immersion after activation
Handled in body, crosslinking agent, the reaction dissolvent of initiator, deoxidation, react synthetic molecules blotting membrane；The selection of the template molecule is former
It is and testing molecule similar substance then；The reaction dissolvent is chloroform and/or methanol；
3) template molecule blotting membrane is immersed in eluting solvent, removes template molecule；
4) molecular engram film after template molecule will be removed to add after testing sample, taken out, using molecular engram film electron spray electricity
Detected from device.
5. method as claimed in claim 4, it is characterised in that the microfiltration membranes are pvdf membrane, PTFE film or cellulose membrane.
6. method as claimed in claim 4, it is characterised in that step 2) in, function monomer is MAA, and crosslinking agent is EGDMA,
Initiator is AIBN；Reaction dissolvent is chloroform and/or methanol.
7. method as claimed in claim 4, it is characterised in that step 2) in, concrete operations are：Fully dissolved with reaction dissolvent
Template molecule, then adds function monomer, prepolymerization 1h；It is subsequently added into add after crosslinking agent and initiator, dissolving and is repaiied through surface
1-3h is reacted in decorations and the microfiltration membranes of activation, deoxidation at room temperature.
8. the method as described in claim 4-7 is any, it is characterised in that step 2) react at room temperature after 1-3h, in addition to
Reacted microfiltration membranes are transferred to 50-70 DEG C of reaction 24-30h of constant temperature under nitrogen environment；After reaction terminates, 10%-20% is used
Acetic acid methanol solution cyclic washing, then with pure water to neutrality, washed and volatilized standby with acetone.
9. any described methods of claim 4-8 are in food security, blood concentration, antibiotic residue or Detecting Pesticide
Application.
10. it is a kind of suitable for molecular engram film electrospray ionization mass spectrometry detect molecular engram film, it is characterised in that by with
Lower section method is prepared：Microfiltration membranes after surface modification and activation are added containing template molecule, function monomer, crosslinking agent, drawn
Send out in the reaction dissolvent of agent and handle, synthetic molecules blotting membrane is reacted in deoxidation；The selection principle of the template molecule be and to be measured point
Sub- similar substance；The reaction dissolvent is chloroform and/or methanol, and the function monomer is MAA, and crosslinking agent is EGDMA, initiator
For AIBN；
Reaction dissolvent is chloroform and/or methanol；
Concrete operations are：Template molecule is fully dissolved with reaction dissolvent, function monomer, prepolymerization 1h is then added；It is subsequently added into
Crosslinking agent and initiator, add the microfiltration membranes of surface modification and activation after dissolving, 1-3h is reacted in deoxidation at room temperature, will be anti-
Microfiltration membranes after answering are transferred to 50-70 DEG C of reaction 24-30h of constant temperature under nitrogen environment；After reaction terminates, 10%-20% acetic acid is used
Methanol solution cyclic washing, then with pure water to neutrality, washed and volatilized with acetone, it is standby.
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| CN114236006A (en) * | 2021-12-17 | 2022-03-25 | 河南省商业科学研究所有限责任公司 | Method for detecting clenbuterol in animal urine |
| CN114236005A (en) * | 2021-12-17 | 2022-03-25 | 河南省商业科学研究所有限责任公司 | Method for detecting clenbuterol in animal food |
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| CN111443152A (en) * | 2020-03-26 | 2020-07-24 | 中国检验检疫科学研究院 | Method and kit for detecting content of quinolone compounds |
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| CN112067685A (en) * | 2020-09-11 | 2020-12-11 | 兰州市食品药品检验所 | Method for rapidly detecting clenbuterol in meat through FaPEx-TD-ESI-MS/MS |
| CN112067685B (en) * | 2020-09-11 | 2023-12-15 | 兰州市食品药品检验所 | Method for rapidly detecting clenbuterol in meat by Fapex-TD-ESI-MS/MS |
| CN114236006A (en) * | 2021-12-17 | 2022-03-25 | 河南省商业科学研究所有限责任公司 | Method for detecting clenbuterol in animal urine |
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