CN106568831A - Porous membrane material for detecting phosphoinositide, and preparation and detection method thereof - Google Patents
Porous membrane material for detecting phosphoinositide, and preparation and detection method thereof Download PDFInfo
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Abstract
The invention provides a porous membrane material for detecting phosphoinositide, and a preparation and detection method thereof. The porous membrane material comprises a porous membrane and a dual-component copolymer grafted to the channels of the porous membrane. The dual-component copolymer is grafted to channels of the porous membrane through the atom transfer free radical polymerization reaction mechanism. The detection method comprises the following steps: putting the porous membrane material between electrochemical cell clamps, injecting electrolyte, measuring the trans-membrane current (I0) under a preset voltage; removing the electrolyte, adding electrolyte containing phosphoinositide, measuring the trans-membrane current (I1) under the preset voltage; according to a formula (I0-I1)/I0, and calculating the value of changed current ratio under the preset voltage, wherein if the value is greater than 0.05, the sample contains phosphoinositide. The method of using provided porous membrane material to detect phosphoinositide has the advantages of high detection sensitivity, quick detection speed, simple operation, and low cost.
Description
Technical field
The present invention relates to bioassay technique field, more particularly to a kind of porous film material for detecting phosphoinositide and
It is prepared and detection method.
Background technology
Phosphoinositide family is closely related with the control of cell behavior as signaling molecule important in eukaryotic cell.Phosphorus
Sour inositol can produce second message,second messenger as signal precursor under rule-activator effect, and itself also can be straight as lipid second message,second messenger
Connect participation many cellular activity such as ion transport, vesicular traffic, signal transduction, cytoskeletoies to assemble again and nuclear gene regulation
Deng.Therefore, the dynamic monitoring and quantitative study of biological internal phosphoinositide are become to the hot issue in life sciences.Mesh
It is front for the detection of phosphoinositide, the main method that adopts be using radioactive element such as32P or3H is during phosphoinositide metabolism
It is marked, first carries out chromatographic isolation, then carries out qualitative or quantitative detection by radiating collection of illustrative plates or mass-spectrometric technique.So
And, said method haves the shortcomings that to waste time and energy, and due to the sensitivity of living things system complex detection it is not high, in addition with radioactivity
It is dangerous.Therefore, when we pay close attention to the concentration rather than concrete structure of its dynamic change in research process, the method
Just it is difficult to meet the requirement of quick detection.In addition, rapidly differentiate the inositol molecule of different degrees of phosphorylation, and it is different with dividing
Structure body is also a great problem.In sum, exploitation has highly sensitive sensor to the phosphoinositide of trace in organism, right
Have great significance in the composition and mechanism of further research intracellular signaling pathway.
The content of the invention
The present invention provides a kind of porous film material for detecting phosphoinositide and its preparation to solve above-mentioned technical problem
With detection method.The material is capable of achieving the hypersensitivity detection and the differentiation of variety classes phosphoinositide of phosphoinositide.
The object of the invention is realized using following proposal：
A kind of porous film material for detecting phosphoinositide, the porous film material includes perforated membrane and is grafted to described
Bi-component copolymer in the duct of perforated membrane, the molecular structure of the bi-component copolymer is as follows：
The perforated membrane is anodic alumina films, polyethylene terephthalate film, polycarbonate membrane, polymethylacrylic acid
Methyl ester film or silicon nitride film.
In such scheme, the perforated membrane be anodic alumina films, aperture 20-100nm.
In such scheme, the preparation method is using atom transition free radical polymerization reaction mechanism, by bi-component copolymer
It is grafted in the duct of perforated membrane, concrete preparation method is as follows：
1) N-isopropylacrylamide monomer and 4- (3- Allyl thiourea bases) benzoic acid list are sequentially added in reaction vessel
Body, both mass ratioes are 1-10:1, it is subsequently adding 30-300mL N, N '-dimethyl formamide solution；
2) catalyst and part are added under anaerobic, in the perforated membrane immersion previous solu that bromination was processed, protect
Nitrogen atmosphere is held, under the conditions of 60-100 DEG C of constant temperature atom transition free radical polymerization reaction 4-25h is carried out；
3) after reaction terminates, wash and be dried, obtain the porous film material.
In such scheme, comprise the following steps：
1) porous film material is put between electrochemical cell fixture, electrolyte is injected in pond, under measurement predetermined voltage
Transmembrane current I0；
2) remove the electrolyte in electrochemical cell, then add may the actual sample containing phosphoinositide, the actual sample
In product addition with step 1) same amount electrolyte, measure predetermined voltage under transmembrane current I1；
3) according to (I0-I1)/I0The numerical value of the curent change ratio being calculated under predetermined voltage, if curent change ratio
The numerical value of example is more than 0.01, then can determine whether to contain phosphoinositide in sample.
In such scheme, also included using activation before the step of being put into porous film material between electrochemical cell fixture
The step of liquid balance activation porous film material.
In such scheme, the activating solution is the Sodium Chloride or Klorvess Liquid that pH=2-5 contains 0.05-2.0mol/L,
Wherein solvent is deionized water.
In such scheme, step 1) and step 2) in electrolyte pH=2-10.
In such scheme, the electrode of the electrochemical cell is silver-silver chloride electrode, hydrargyrum-mercuric chloride or graphite electrode.
In such scheme, the step 1) and step 2) electrolyte be the Sodium Chloride containing 0.05-2.0mol/L or chlorine
Change potassium solution, solvent is 0 for the volume ratio of acetonitrile/deionized water solution, the acetonitrile and deionized water:100-50:50.
In such scheme, concretely comprising the following steps for transmembrane current is measured：Gathered using picoammeter, power supply is applied at electrode two ends
Plus -0.2 to+0.2V pulse voltage, each 1-10 second pulse voltage persistent period.
The present invention will have in the polymer-modified duct to perforated membrane of response to phosphoinositide, to build bionical people
Work ion channel.When phosphoinositide passes through nanochannel, and the combination of the polymer of inner surfaces of pores modification causes the latter's conformation
Generation acute variation, so as to cause duct effective diameter to diminish, is finally realized by the change of picoammeter measuring holes electric current
The ultrasensitiveness of different degrees of polyphosphorylated inositol molecule is detected.
Beneficial effects of the present invention are：
1st, porous film material prepared by the present invention is high with detection sensitivity when phosphoinositide is detected, detection speed is fast,
Advantage easy to operate, with low cost, is very suitable for the dynamic monitoring of phosphoinositide contents level in complex sample system；
2nd, porous film material prepared by the present invention is different to different phosphoinositide responses when phosphoinositide is detected,
Therefore the differentiation of different phosphoinositides can be realized；
3rd, when phosphoinositide is detected, detection signal is micro-current to porous film material prepared by the present invention, is easily controlled, supervises
Survey and change into other signals, the system compatibility is good, easily with other devices or Instrument crosslinking, with good extensibility.
Description of the drawings
Fig. 1 is the schematic arrangement of bi-component copolymer.
Fig. 2 is the structural representation of porous film material.
Fig. 3 is anodised aluminium environmental microbes SEM figures.
Fig. 4 is to be grafted the anodised aluminium environmental microbes SEM figures after bi-component copolymer.
Fig. 5 be bi-component copolymer surface to (1,3) IP2, (1,3,5) InsP3 and inositol hexaphosphate
Crystal microbalance (QCM) adsorption curve.
Fig. 6 is bi-component copolymer surface atom force microscope shape appearance figure.
Fig. 7 is that the atomic force microscope shape appearance figure after inositol hexaphosphate is soaked on bi-component copolymer surface.
Fig. 8 is schematic device of the porous film material when phosphoinositide is detected.
Fig. 9 is to be grafted the anodic alumina films VA characteristic curve after bi-component copolymer.
Figure 10 is that variable concentrations (1,3) IP2 stimulates lower cross-film micro-current change curve.
Figure 11 is that variable concentrations (1,3,5) InsP3 stimulates lower cross-film micro-current change curve.
Figure 12 is that variable concentrations inositol hexaphosphate stimulates lower cross-film micro-current change curve.
Figure 13 is that vitamin C, glycine, carbamide, glucose and the inositol hexaphosphate of 1nM stimulates lower cross-film micro-current change
Ratio comparison diagram.
Specific embodiment
To make present disclosure, technical scheme and advantage become more apparent, below in conjunction with specific embodiments and the drawings
The present invention is expanded on further, these embodiments are merely to illustrate the present invention, and the present invention is not limited only to following examples.
Raw materials used and equipment in embodiment:
Various perforated membranes are buied by Shenzhen topology Jing Mo Science and Technology Ltd.s.(99.999%) CuBr, joins pyrrole to cuprous bromide
Pyridine class part, organic base, N-isopropylacrylamide, acryloyl chloride, para-amino benzoic acid and test with various phosphoinositides by
Sigma-Aldrich companies buy.Acetone, methanol, N, N '-dimethyl Methanamide (DMF) is buied by Alpha Co., Ltd.Isopropyl
Acrylamide is placed on standby in vacuum desiccator using front use normal hexane recrystallization three times.Other reagents are using commercially available
Analysis is pure.Crystal microbalance (QCM) adsorpting data is obtained by Q-Sense E4system detections.Atomic force microscope topographic data
Obtained by types AFM of Bruker Multimode 8.PSTM topographic data is gathered by Hitachi S-4800 types SEM
Obtain.Cross-film micro-current data are by the type picoammeter automatic data collections of Keithley 6487 and record.
Embodiment 1
The preparation of the porous film material of grafting bi-component copolymer
The structure of double-component copolymer is as shown in figure 1, wherein X=0.01-0.5.By taking X=0.2 as an example, in tri- mouthfuls of 100mL
4.8mmol N-isopropylacrylamides (NIPAM) and 1.2mmol 4- (3- Allyl thiourea bases) benzoic acid are added in flask
(ATBA), and 60mL N, N '-dimethyl Methanamide (DMF) ultrasonic dissolution 10 minutes are added.After logical nitrogen 20 minutes, add
32mg cuprous bromides (CuBr) powder, mix homogeneously.The perforated membrane that bromination was processed is added in flask, and following reaction system is taken out
Vacuum-inflated with nitrogen, removes oxygen remaining in reaction system.Then 0.16mL N, the first of N, N', N', N'- five are added by injection
Base diethylenetriamines (PMDETA) or bipyridine ligand, then carry out again a deoxidation treatment.In nitrogen protection, 70 DEG C of constant temperature
Under the conditions of, take out after reaction 15h, respectively with 100mL DMF and deionized water soaking flushing in order, with nitrogen standby is dried up
With obtaining porous film material as shown in Figure 2.
The perforated membrane is anodic alumina films, polyethylene terephthalate film, polycarbonate membrane, poly-methyl methacrylate
Ester film or silicon nitride film.By taking anodic alumina films (hereinafter referred to as PAA films) as an example, Fig. 3 and Fig. 4 is porous material before and after grafting
Surface SEM schemes.
Preliminary Applications example
Embodiment 2
By QCM-D adsorbance methods for measuring, with evaluate the double-component copolymer surface to (1,3) IP2,
(1,3,5) the InsP3 absorption behavior different with inositol hexaphosphate.Similar approach is total to the double-component as described in embodiment 1
Polymers is grafted to QCM-D chip surfaces, is respectively 1 μ g/mL to concentration by carrier fluid of deionized water under the conditions of 20 DEG C of temperature control
(1,3) IP2, (1,3,5) InsP3 and inositol hexaphosphate carry out adsorption experiment.Fig. 5 shows that the double-component is total to
Polymer surface to (1,3) IP2, (1,3,5) InsP3 and inositol hexaphosphate have stronger absorption and have
Adsorbance of kind of the phosphoinositide of certain separating capacity, i.e., three on the double-component copolymer surface is different.
Embodiment 3
What is prepared as described in embodiment 2 is grafted the thin-film material that double-component copolymer is obtained, immersion on QCM-D chips
20 minutes in 10ml inositol hexaphosphate containing 10mg aqueous solution.Afterwards by afm scan pattern observation six phosphorus of immersion
The change of the double-component copolymer film material surface before and after sour inositol.The polymer thin membrane material is observed that by Fig. 6 and Fig. 7
Material there occurs obvious morphology change in surface after immersion inositol hexaphosphate solution, illustrate the thin polymer film to inositol hexaphosphate
With response, the change of macroeffect can be amplified to.
Embodiment 4
By the porous material of double-component copolymer modification by taking PAA films as an example, its activation equilibrium process is：Will be blank or double
The PAA films of component copolymer grafted are fitted in surface plate, add activating solutions of the 200 μ L pH=2.5 containing 0.1mol/L Sodium Chloride,
After standing 10 minutes, PAA films are taken out, after being dried up with nitrogen after 200 μ L deionized water rinsings following experimental implementation are carried out.
The PAA films of the blank or double-component copolymer modification after above-mentioned activation are put between electrochemical cell fixture, so
Electrolyte of the pH=7 containing 0.1mol/L Sodium Chloride is injected in backward pond, vibration to be excluded and stand 5 minutes after film blibbing, electrification
Learn and measure its transmembrane current with picoammeter after pond two ends insertion silver-silver chloride electrode (concrete device is as shown in Figure 8).Measure its across
Membrane current is gathered using picoammeter, makes power supply apply the pulse voltage of -0.2～+0.2V, each arteries and veins at electrode two ends during collection
Rush voltage-duration 4 seconds, and corresponding cross-film micro-current is recorded on connected computer by picoammeter automatically.
As seen from Figure 9, before and after grafting double-component copolymer, change there occurs by the ion rate travel of PAA films, its
VA characteristic curve there occurs significant change, and electric current is presented downward trend, illustrate the electricity for being grafted the PAA films after double-component copolymer
Chemical response signal enhancing.
Embodiment 5
The present invention carries out the detection of phosphoinositide using the PAA films after grafting double-component copolymer, comprises the following steps that：
1) porous film material after grafting double-component copolymer is put between electrochemical cell fixture, pH=is injected in pond
7 electrolyte containing 0.1mol/L Sodium Chloride, measure the transmembrane current I of+2V0；
2) electrolyte in electrochemical cell is removed, then is added containing 10-9Electrolyte (the pH of mol/L (1,3) IP2
=7, Sodium Chloride containing 0.1mol/L), vibration to be excluded and stand 5 minutes after film blibbing, electrochemical cell two ends insertion silver-silver chloride
The transmembrane current I of+2V is measured after electrode with picoammeter1；
3) according to (I0-I1)/I0It is calculated the numerical value of the curent change ratio that voltage is+2V.
4) with above-mentioned steps 1) to 3) roughly the same, difference is by step 2) in (1,3) IP2
Concentration is substituted for respectively 10-8、10-7、10-6With 10-5Mol/L, can obtain the numerical value of four additional curent change ratio.Utilize
Above-mentioned same method, is only substituted for the perforated membrane without graft polymers by the porous film material after grafting double-component copolymer
(blank group), can obtain the numerical value of five curent change ratios.
So that (1,3) concentration of IP2 is abscissa, and the perforated membrane curent change ratio before and after grafting is vertical coordinate
Curve is drawn, as shown in Figure 10.From fig. 10 it can be seen that for the impact and less of blank PAA films, and for being grafted with double groups
The PAA films of part copolymer, then there occurs the phenomenon that in various degree significantly electric current declines.As can be seen here, the double-component copolymer
The PAA materials of grafting for (1,3) IP2 show the different responses for depending on its concentration, for concentration is
10-9(1,3) IP2 of mol/L can accomplish highly sensitive detection.
Embodiment 6
The present embodiment is roughly the same with embodiment 5, and difference is, in embodiment 5 (1,3) IP2 replace
For (1,3,5) InsP3, as a result as shown in figure 11.It is as seen from Figure 11, simultaneously little for the impact of blank PAA films,
And for the PAA films of double-component copolymer are grafted with, then there occurs the phenomenon that in various degree significantly electric current declines.Thus may be used
See, the PAA materials of the double-component copolymer grafted for (1,3,5) InsP3 show the difference for depending on its concentration
Response；And comparing, (1,3) IP2 is 10 in concentration-9During mol/L, (1,3,5) InsP3 cause cross-film
Electric current has decline by a larger margin.
Embodiment 7
The present embodiment is roughly the same with embodiment 5, and difference is, by embodiment 5 (1,3) IP2 replace
Inositol hexaphosphate is changed to, as a result as shown in figure 12.As seen from Figure 12, the impact of blank PAA films is simultaneously little, and for grafting
There are the PAA films of double-component copolymer, then there occurs the phenomenon that in various degree significantly electric current declines.As can be seen here, the double-component
The PAA materials of copolymer grafted depend on the different responses of its concentration for inositol hexaphosphate shows；And compare
(1,3) IP2 or (1,3,5) InsP3, concentration be 10-9During mol/L, inositol hexaphosphate causes cross-film electricity
Stream has decline by a larger margin.
Therefore, porous film material highly sensitive response for phosphoinositide has of the invention, test limit reaches 10-9mol/L-
10-8mol/L。
Embodiment 8
According to the methods described of embodiment 5, by step 2) in contain in electrolyte (1,3) IP2 change into respectively
The vitamin C of 1nM concentration, glycine, carbamide, glucose, carry out transmembrane current test.
As shown in figure 13, as a example by the cross-film micro-current equally when+0.2V, 1nM concentration is added respectively in toward electrolyte
Vitamin C, glycine, carbamide or glucose after, the impact of the PAA films for blank PAA films or after being grafted is all
Less, curent change ratio adds six phosphorus of 1nM concentration less than 0.01, and before as described in Example 7 in toward electrolyte
During sour inositol；Significantly decline is there occurs by being grafted with the electric current of the PAA films of double-component copolymer, curent change ratio reaches
0.14 or so, far above electricity of the Common materials as caused by vitamin C, glycine, carbamide, glucose etc. in other biological body
Rheology ratio.As can be seen here, the PAA materials of the double-component copolymer grafted have gone out specificity for phosphoinositide material exhibits
Response, not by the Interference Detection of internal other common biomolecule.
Embodiment 9
According to the methods described of embodiment 5, the phosphoinositide in actual sample is tested,
1) porous film material after grafting double-component copolymer is put between electrochemical cell fixture, pH=is injected in pond
7 electrolyte containing 0.1mol/L Sodium Chloride, measure the transmembrane current I of+2V0；
2) electrolyte in electrochemical cell is removed, then adds actual sample, actual sample pH=7, chlorination containing 0.1mol/L
The electrolyte of sodium, vibration to be excluded and stand 5 minutes after film blibbing, and after electrochemical cell two ends insertion silver-silver chloride electrode skin is used
The transmembrane current I of peace measurement amount+2V1；
3) according to (I0-I1)/I0The numerical value of the curent change ratio being calculated under predetermined voltage, if curent change ratio
The numerical value of example is more than 0.05, then can determine whether to contain phosphoinositide in sample.The calculated numerical value of the present embodiment is 0.07, then may be used
Contain phosphoinositide in judgement sample.
In sum, the porous film material of double-component copolymer modification of the present invention respond well energy for phosphoinositide has
Power, by the monitoring of transmembrane current the preliminary detection by quantitative to phosphoinositide can be realized, and to different phosphoinositide exhibitions
Certain separating capacity is revealed.Simultaneously with detection speed is fast, sensitivity is high and low cost compared with traditional detection method
Honest and clean advantage.Therefore extensive, high flux, high-precision phosphoinositide concentration inspection can be applied in complex biological system
Survey, be the conventional signal of telecommunication additionally, due to the method detection signal, compatibility is good, therefore is expected to combine other detection meanss, in phosphorus
The research field of the detection and analysis of sour inositol or even biological signal path in vivo plays unique effect.
Claims (10)
1. a kind of porous film material for detecting phosphoinositide, it is characterised in that the porous film material include perforated membrane and
The bi-component copolymer being grafted in the duct of the perforated membrane, the molecular structure of the bi-component copolymer is as follows：
The perforated membrane is anodic alumina films, polyethylene terephthalate film, polycarbonate membrane, polymethyl methacrylate
Film or silicon nitride film.
2. porous film material as claimed in claim 1, it is characterised in that the perforated membrane is anodic alumina films, aperture 20-
100nm。
3. the preparation method of porous film material as claimed in claim 1, it is characterised in that the preparation method is turned using atom
Raolical polymerizable mechanism is moved, by the duct of bi-component copolymer grafted to perforated membrane, concrete preparation method is as follows：
1) N-isopropylacrylamide monomer and 4- (3- Allyl thiourea bases) benzoic acid monomer are sequentially added in reaction vessel, two
Person's mass ratio is 1-10:1, it is subsequently adding 30-300mL N, N '-dimethyl formamide solution；
2) catalyst and part are added under anaerobic, in the perforated membrane immersion previous solu that bromination was processed, keep nitrogen
Gas atmosphere, under the conditions of 60-100 DEG C of constant temperature atom transition free radical polymerization reaction 4-25h is carried out；
3) after reaction terminates, wash and be dried, obtain the porous film material.
4. the detection method of the as claimed in claim 1 porous film material for being used to detect phosphoinositide, it is characterised in that include
Following steps：
1) porous film material is put between electrochemical cell fixture, electrolyte is injected in pond, measure the cross-film under predetermined voltage
Electric current I0；
2) remove the electrolyte in electrochemical cell, then add may the actual sample containing phosphoinositide, in the actual sample
Addition with step 1) same amount electrolyte, measure predetermined voltage under transmembrane current I1；
3) according to (I0-I1)/I0The numerical value of the curent change ratio being calculated under predetermined voltage, if curent change ratio
Numerical value is more than 0.05, then can determine whether to contain phosphoinositide in sample.
5. detection method as claimed in claim 4, it is characterised in that be put into porous film material between electrochemical cell fixture
The step of before also include using activating solution balance activation porous film material the step of.
6. detection method as claimed in claim 5, it is characterised in that the activating solution is that pH=2-5 contains 0.05-2.0mol/L
Sodium Chloride or Klorvess Liquid, wherein solvent be deionized water.
7. detection method as claimed in claim 4, it is characterised in that step 1) and step 2) in electrolyte pH=2-
10。
8. detection method as claimed in claim 4, it is characterised in that the electrode of the electrochemical cell be silver-silver chloride electrode,
Hydrargyrum-mercuric chloride or graphite electrode.
9. detection method as claimed in claim 4, it is characterised in that the step 1) and step 2) electrolyte be containing
The Sodium Chloride or Klorvess Liquid of 0.05-2.0mol/L, solvent is acetonitrile/deionized water solution, the acetonitrile and deionization
The volume ratio of water is 0:100-50:50.
10. detection method as claimed in claim 4, it is characterised in that measurement transmembrane current is concretely comprised the following steps：Using pico-ampere
Meter collection, power supply applies -0.2 to+0.2V pulse voltage, each 1-10 second pulse voltage persistent period at electrode two ends.
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| CN111410768A (en) * | 2020-03-07 | 2020-07-14 | 武汉理工大学 | Three-component intelligent polymer modified porous membrane material and preparation method and application thereof |
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