CN106568831B - A kind of porous film material and its preparation and detection method for detecting phosphoinositide - Google Patents

Abstract

The present invention provides a kind of porous film material for detecting phosphoinositide and its preparation and detection method, the porous film material include perforated membrane and the bi-component copolymer being grafted in the duct of perforated membrane.The porous film material is to utilize atom transition free radical polymerization reaction mechanism, will be in the duct of bi-component copolymer grafted to perforated membrane.Using the material, detection method includes the following steps: porous film material being put between electrochemical cell fixture, electrolyte is injected, measures the transmembrane current I under predetermined voltage₀；Electrolyte is removed, then adds the electrolyte containing phosphoinositide, measures the transmembrane current I under predetermined voltage₁；According to (I₀‑I₁)/I₀The numerical value of the curent change ratio under predetermined voltage is calculated, if the numerical value of curent change ratio is greater than 0.05, can determine whether to contain phosphoinositide in sample.Porous film material prepared by the present invention has detection sensitivity high when detecting phosphoinositide, detects fast, easy to operate, the low in cost advantage of speed.

Description

A kind of porous membrane material for detecting phosphoinositide and preparation and detection method thereof

technical field

The invention relates to the technical field of biological analysis, in particular to a porous membrane material for detecting phosphoinositide and a preparation and detection method thereof.

Background technique

As important signaling molecules in eukaryotic cells, the phosphoinositide family is closely related to the control of cell behavior. Phosphoinositide, as a signal precursor, can generate second messengers under the action of agonists, and itself can directly participate in many cellular activities as lipid second messengers such as ion transport, vesicle transport, signal transduction, cytoskeleton reassembly and nuclear gene regulation. Therefore, the dynamic monitoring and quantitative research of phosphoinositide in vivo has become a hot issue in life sciences. At present, for the detection of phosphoinositide, the main method is to use radioactive elements such as ³² P or ³ H to label in the process of phosphoinositide metabolism, first perform chromatographic separation, and then perform qualitative or quantitative detection by radiographic or mass spectrometry. . However, the above method has the disadvantage of being time-consuming and labor-intensive, and due to the complex biological system, the detection sensitivity is not high, and in addition, it has the danger of radioactivity. Therefore, when we focus on the dynamic concentration rather than the specific structure in the research process, this method is difficult to meet the requirements of rapid detection. In addition, the rapid identification of differently phosphorylated inositol molecules and their isomers is also a major challenge. In summary, the development of sensors with high sensitivity to trace amounts of phosphoinositide in vivo is of great significance for further research on the composition and mechanism of intracellular signaling pathways.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a porous membrane material for detecting phosphoinositide and a preparation and detection method thereof. The material can achieve ultra-high sensitivity detection of phosphoinositide and the discrimination of different kinds of phosphoinositide.

The object of the present invention adopts the following scheme to realize:

A porous membrane material for detecting phosphoinositide, the porous membrane material comprises a porous membrane and a two-component copolymer grafted into the pores of the porous membrane, and the molecular structure of the two-component copolymer is as follows :

The porous film is an anodic aluminum oxide film, a polyethylene terephthalate film, a polycarbonate film, a polymethyl methacrylate film or a silicon nitride film.

In the above solution, the porous film is an anodic aluminum oxide film with a pore size of 20-100 nm.

In the above scheme, the preparation method is to use the atom transfer radical polymerization reaction mechanism to graft the two-component copolymer into the pores of the porous membrane, and the specific preparation method is as follows:

1) in the reaction vessel, add isopropylacrylamide monomer and 4-(3-propenyl thioureido) benzoic acid monomer successively, and the mass ratio of the two is 1-10:1, then add 30-300mL N, N'-dimethylformamide solution;

2) Add catalyst and ligand under anaerobic conditions, immerse the brominated porous membrane in the aforementioned solution, maintain a nitrogen atmosphere, and carry out atom transfer radical polymerization at a constant temperature of 60-100°C for 4-25h;

3) After the reaction, wash and dry to obtain the porous membrane material.

The above scheme includes the following steps:

1) put the porous membrane material between the electrochemical cell fixtures, inject electrolyte in the cell, and measure the transmembrane current I ₀ under a predetermined voltage;

2) removing the electrolyte in the electrochemical cell, then adding an actual sample that may contain phosphoinositide, adding the electrolyte with the same content as in step 1) in the actual sample, and measuring the transmembrane current I ₁ under a predetermined voltage;

3) Calculate the value of the current change ratio under the predetermined voltage according to (I ₀ -I ₁ )/I ₀ , if the value of the current change ratio is greater than 0.01, it can be determined that the sample contains phosphoinositide.

In the above solution, before the step of placing the porous membrane material between the clamps of the electrochemical cell, the step of balancing the activation of the porous membrane material with an activation solution is included.

In the above scheme, the activation solution is a sodium chloride or potassium chloride solution with pH=2-5 containing 0.05-2.0 mol/L, wherein the solvent is deionized water.

In the above scheme, the pH of the electrolyte in step 1) and step 2) is 2-10.

In the above solution, the electrodes of the electrochemical cell are silver-silver chloride electrodes, mercury-mercury chloride electrodes or graphite electrodes.

In the above-mentioned scheme, the electrolyte solution of described step 1) and step 2) is the sodium chloride or potassium chloride solution containing 0.05-2.0mol/L, and the solvent is acetonitrile/deionized water solution, and the acetonitrile and deionized water The volume ratio is 0:100-50:50.

In the above scheme, the specific steps for measuring the transmembrane current are as follows: using a picoammeter for acquisition, the power supply applies a pulse voltage of -0.2 to +0.2V at both ends of the electrode, and the duration of each pulse voltage is 1-10 seconds.

The present invention modifies the polymer responsive to phosphoinositide into the pores of the porous membrane to construct a biomimetic artificial ion channel. When phosphoinositide passes through the nanochannel, the combination with the polymer modified on the inner surface of the channel causes the latter to undergo a drastic change in the conformation, resulting in the reduction of the effective diameter of the channel. Ultrasensitive detection of myo-inositol molecules.

The beneficial effects of the present invention are:

1. When detecting phosphoinositide, the porous membrane material prepared by the present invention has the advantages of high detection sensitivity, fast detection speed, simple operation and low cost, and is very suitable for dynamic monitoring of the content level of phosphoinositide in complex sample systems;

2. The porous membrane material prepared by the present invention has different responsiveness to different phosphoinositols when detecting phosphoinositol, so it can realize the distinction of different phosphoinositols;

3. When the porous membrane material prepared by the present invention detects phosphoinositide, the detection signal is microcurrent, which is easy to control, monitor and convert into other signals. The system has good compatibility and is easy to be combined with other devices or instruments. of scalability.

Description of drawings

Figure 1 is a schematic diagram of the molecular structure of the two-component copolymer.

FIG. 2 is a schematic diagram of the structure of the porous membrane material.

Figure 3 is the SEM image of the surface morphology of the anodic aluminum oxide film.

Figure 4 is a SEM image of the surface morphology of the anodic aluminum oxide film after grafting the bicomponent copolymer.

Figure 5 is a quartz microbalance (QCM) adsorption curve of (1,3) inositol diphosphate, (1,3,5) inositol triphosphate and inositol hexaphosphate on the surface of the two-component copolymer.

Figure 6 is an atomic force microscope topography of the surface of the bicomponent copolymer.

Figure 7 is an atomic force microscope topography of the surface of the two-component copolymer after soaking inositol hexaphosphate.

FIG. 8 is a schematic diagram of the device for the detection of phosphoinositide by the porous membrane material.

Fig. 9 is the voltammetric characteristic curve of the anodic aluminum oxide film after grafting the two-component copolymer.

Figure 10 is the change curve of transmembrane microcurrent under the stimulation of different concentrations of (1,3) inositol bisphosphate.

Figure 11 is the change curve of transmembrane microcurrent under the stimulation of inositol triphosphate with different concentrations (1,3,5).

Figure 12 is the change curve of transmembrane microcurrent under the stimulation of different concentrations of inositol hexaphosphate.

Fig. 13 is a comparison chart of the change ratio of transmembrane microcurrent under the stimulation of 1 nM vitamin C, glycine, urea, glucose and inositol hexaphosphate.

Detailed ways

In order to make the content, technical solutions and advantages of the present invention clearer, the present invention will be further described below with reference to specific embodiments and accompanying drawings. These embodiments are only used to illustrate the present invention, and the present invention is not limited to the following embodiments.

Raw materials and equipment used in the embodiment:

Various porous membranes were purchased from Shenzhen Topology Membrane Technology Co., Ltd. Cuprous bromide (CuBr, 99.999%), bipyridine ligands, organic bases, isopropylacrylamide, acryloyl chloride, para-aminobenzoic acid and various phosphoinositols for testing were purchased from Sigma-Aldrich Company. Acetone, methanol, and N,N'-dimethylformamide (DMF) were purchased from Alpha Corporation. Isopropylacrylamide was recrystallized three times with n-hexane before use, and placed in a vacuum desiccator for later use. All other reagents were of commercially available analytical grade. Quartz microbalance (QCM) adsorption data were obtained by Q-Sense E4system detection. Atomic force microscopy topography data were obtained with a Bruker Multimode Model 8 AFM. Scanning tunneling microscope topography data were acquired by Hitachi S-4800 SEM. Transmembrane microcurrent data were automatically collected and recorded by a Keithley Model 6487 picoammeter.

Example 1

Preparation of Porous Membrane Materials Grafted with Bicomponent Copolymers

The structure of the two-component copolymer is shown in Figure 1, where X=0.01-0.5. Taking X=0.2 as an example, add 4.8mmol isopropylacrylamide (NIPAM) and 1.2mmol 4-(3-propenylthioureido)benzoic acid (ATBA) to a 100mL three-necked flask, and add 60mL N,N' - Dimethylformamide (DMF) sonicated for 10 minutes. After 20 minutes of nitrogen flow, 32 mg of cuprous bromide (CuBr) powder was added and mixed uniformly. The brominated porous membrane was added to the flask, and then the reaction system was evacuated and filled with nitrogen to remove residual oxygen in the reaction system. Then 0.16 mL of N,N,N',N',N'-pentamethyldiethylenetriamine (PMDETA) or bipyridine ligand was added by injection, followed by one more deoxygenation treatment. Under the conditions of nitrogen protection and constant temperature of 70 °C, take out after 15 h of reaction, soak and rinse with 100 mL of DMF and deionized water in sequence, dry with nitrogen for use, and obtain the porous membrane material shown in Figure 2.

The porous film is an anodic aluminum oxide film, a polyethylene terephthalate film, a polycarbonate film, a polymethyl methacrylate film or a silicon nitride film. Taking the anodic aluminum oxide film (hereinafter referred to as PAA film) as an example, Fig. 3 and Fig. 4 are the surface SEM images of the porous material before and after grafting.

Preliminary application example

Example 2

The different adsorption behaviors of (1,3) inositol diphosphate, (1,3,5) inositol triphosphate and inositol hexaphosphate on the surface of the two-component copolymer were evaluated by the method of QCM-D adsorption capacity determination. The two-component copolymer was grafted to the surface of the QCM-D chip according to the method similar to that described in Example 1. Under the condition of temperature control at 20°C, deionized water was used as the carrier liquid, and the concentration of 1 μg/mL (1, 3) Inositol diphosphate, (1,3,5) inositol triphosphate and inositol hexaphosphate were used for adsorption experiments. Figure 5 shows that the surface of the two-component copolymer has strong adsorption to (1,3) inositol diphosphate, (1,3,5) inositol triphosphate and inositol hexaphosphate, and has a certain ability to distinguish, That is, the adsorption amounts of the three inositol phosphates on the surface of the two-component copolymer are different.

Example 3

The film material prepared by grafting the two-component copolymer on the QCM-D chip prepared as described in Example 2 was soaked in 10 ml of an aqueous solution containing 10 mg of inositol hexaphosphate for 20 minutes. Then, the surface changes of the two-component copolymer film material before and after soaking in inositol hexaphosphate were observed by atomic force microscope scanning mode. From Figure 6 and Figure 7, it can be observed that the surface of the polymer film material has obvious morphology changes after soaking in the inositol hexaphosphate solution, indicating that the polymer film is responsive to inositol hexaphosphate and can be enlarged to Changes in macro effects.

Example 4

Taking the two-component copolymer modified porous material as an example of a PAA film, the activation equilibrium process is as follows: put the blank or two-component copolymer grafted PAA film into a watch glass, add 200 μL pH=2.5 containing 0.1mol/ After standing for 10 minutes with the activation solution of L sodium chloride, the PAA membrane was taken out, rinsed with 200 μL of deionized water, dried with nitrogen, and then the following experimental operations were performed.

Put the above activated blank or two-component copolymer modified PAA membrane between the electrochemical cell fixtures, then inject pH=7 electrolyte containing 0.1mol/L sodium chloride into the cell, shake to remove bubbles on the membrane surface After standing for 5 minutes, silver-silver chloride electrodes were inserted at both ends of the electrochemical cell, and the transmembrane current was measured with a picoammeter (the specific device is shown in Figure 8). To measure the transmembrane current, a picoammeter is used to collect it. During the collection, the power source applies a pulse voltage of -0.2 to +0.2V to both ends of the electrode. The corresponding transmembrane microcurrents were recorded.

As can be seen from Figure 9, before and after the grafting of the bicomponent copolymer, the ion movement rate through the PAA membrane changed, the voltammetric curve of the ion changed significantly, and the current showed a downward trend, indicating that after the grafting of the bicomponent copolymer. The electrochemical response signal of the PAA film is enhanced.

Example 5

The present invention utilizes the PAA film after the grafted two-component copolymer to carry out the detection of phosphoinositide, and the concrete steps are as follows:

1) Put the porous membrane material after grafting the two-component copolymer between the clamps of the electrochemical cell, inject the electrolyte with pH=7 containing 0.1mol/L sodium chloride into the cell, and measure the transmembrane current of +2V I ₀ ;

2) Remove the electrolyte in the electrochemical cell, and then add the electrolyte (pH=7, containing 0.1mol/L sodium chloride) containing 10 ^-9 mol/L (1,3) inositol diphosphate, and shake to remove The membrane surface was left to stand for 5 minutes after air bubbles, and the two ends of the electrochemical cell were inserted into silver-silver chloride electrodes to measure the transmembrane current I ₁ of +2V with a picoammeter;

3) According to (I ₀ -I ₁ )/I ₀ , the value of the current change ratio with a voltage of +2V is obtained.

4) It is roughly the same as the above steps 1) to 3), except that the concentration of (1,3) inositol bisphosphate in step 2) is replaced by ^10-8 , ^10-7 , ^10-6 and 10, respectively ^-5 mol/L, the other four values of the current change ratio can be obtained. Using the same method as above, only replacing the porous membrane material after grafting the bicomponent copolymer with the porous membrane without grafting polymer (blank group), five values of current change ratios can be obtained.

Taking the concentration of (1,3) inositol bisphosphate as the abscissa, and the current change ratio of the porous membrane before and after grafting as the ordinate, a curve was drawn, as shown in Figure 10. It can be seen from Figure 10 that the effect on the blank PAA film is not great, while for the PAA film grafted with the two-component copolymer, the phenomenon of obvious current drop occurs to different degrees. It can be seen that the two-component copolymer-grafted PAA material exhibits different responsiveness to (1,3) ^inositol diphosphate depending on its concentration. 3) Inositol bisphosphate can be detected with high sensitivity.

Example 6

This example is substantially the same as Example 5, except that (1,3) inositol diphosphate in Example 5 is replaced by (1,3,5) inositol triphosphate, and the results are shown in FIG. 11 . It can be seen from Figure 11 that the effect on the blank PAA film is not great, while for the PAA film grafted with the two-component copolymer, the phenomenon of obvious current drop occurs in different degrees. It can be seen that the two-component copolymer-grafted PAA material exhibits different responsiveness to (1,3,5) inositol triphosphate depending on its concentration; and compared with (1,3) inositol triphosphate Alcohol, at a concentration of 10 ^-9 mol/L, (1,3,5) inositol triphosphate led to a greater decrease in transmembrane current.

Example 7

This example is substantially the same as Example 5, except that (1,3) inositol diphosphate in Example 5 is replaced with inositol hexaphosphate, and the results are shown in FIG. 12 . It can be seen from Figure 12 that the effect of the blank PAA film is not great, while for the PAA film grafted with the two-component copolymer, the phenomenon of obvious current drop occurs in different degrees. It can be seen that the two-component copolymer-grafted PAA material exhibits different responsiveness to inositol hexaphosphate depending on its concentration; and compared with (1,3) inositol bisphosphate or (1,3, 5) Inositol triphosphate, when the concentration is 10 ^-9 mol/L, inositol hexaphosphate leads to a greater decrease in transmembrane current.

Therefore, the porous membrane material of the present invention has a highly sensitive response to phosphoinositide, and the detection limit reaches 10 ^-9 mol/L-10 ^-8 mol/L.

Example 8

According to the method described in Example 5, the (1,3) inositol bisphosphate contained in the electrolyte in step 2) was replaced with vitamin C, glycine, urea, and glucose at a concentration of 1 nM, respectively, and a transmembrane current test was performed.

As shown in Figure 13, also taking the transmembrane microcurrent at +0.2V as an example, after adding vitamin C, glycine, urea or glucose at a concentration of 1 nM to the electrolyte, no matter for the blank PAA membrane or the grafted The influence of the PAA film is not large, and the current change ratio is less than 0.01. As previously described in Example 7, when adding 1 nM concentration of inositol hexaphosphate to the electrolyte; The current of the PAA film decreased significantly, and the current change ratio reached about 0.14, which was much higher than that caused by other common substances in organisms such as vitamin C, glycine, urea, and glucose. It can be seen that the PAA material grafted by the two-component copolymer exhibits a specific response to phosphoinositide substances, and is not interfered by other common biomolecules in the body.

Example 9

According to the method described in Example 5, the phosphoinositide in the actual sample was tested,

1) Put the porous membrane material after grafting the two-component copolymer between the clamps of the electrochemical cell, inject the electrolyte with pH=7 containing 0.1mol/L sodium chloride into the cell, and measure the transmembrane current of +2V I ₀ ;

2) Remove the electrolyte in the electrochemical cell, then add the actual sample, the actual sample pH=7, the electrolyte containing 0.1mol/L sodium chloride, shake to remove the bubbles on the surface of the membrane and let it stand for 5 minutes, the electrochemical cell two After the silver-silver chloride electrode is inserted into the end, the transmembrane current I ₁ of +2V is measured with a picoammeter;

3) Calculate the value of the current change ratio under the predetermined voltage according to (I ₀ -I ₁ )/I ₀ , if the value of the current change ratio is greater than 0.05, it can be judged that the sample contains phosphoinositide. If the value calculated in this example is 0.07, it can be judged that the sample contains phosphoinositide.

To sum up, the porous membrane material modified by the two-component copolymer of the present invention has a good response ability to phosphoinositide, and can realize the preliminary quantitative detection of phosphoinositide through the monitoring of transmembrane current, and can detect different phosphoinositides. Inositol exhibits some discriminatory power. At the same time, compared with traditional detection methods, it has the advantages of fast detection speed, high sensitivity and low cost. Therefore, it can be applied to large-scale, high-throughput, and high-precision detection of phosphoinositide concentration in complex biological systems. In addition, because the detection signal of this method is a commonly used electrical signal and has good compatibility, it is expected to combine other detection methods. The detection and analysis of myo-inositol and even the research of in vivo signaling pathways play a unique role.

Claims (9)

1. a preparation method for detecting the porous membrane material of phosphoinositide, it is characterized in that, described porous membrane material comprises porous membrane and the bicomponent copolymer that is grafted in the pore of described porous membrane, described The molecular structure of the two-component copolymer is as follows: The porous film is an anodic aluminum oxide film, a polyethylene terephthalate film, a polycarbonate film, a polymethyl methacrylate film or a silicon nitride film; the preparation method is as follows: 1) in the reaction vessel, add isopropylacrylamide monomer and 4-(3-propenyl thioureido) benzoic acid monomer successively, and the mass ratio of the two is 1-10:1, then add 30-300mL N, N'-dimethylformamide solution; 2) Add catalyst and ligand under anaerobic conditions, immerse the brominated porous membrane in the aforementioned solution, maintain a nitrogen atmosphere, and carry out atom transfer radical polymerization at a constant temperature of 60-100°C for 4-25h; 3) After the reaction, wash and dry to obtain the porous membrane material. 2 . The preparation method according to claim 1 , wherein the porous film is an anodic aluminum oxide film with a pore diameter of 20-100 nm. 3 . 3. the detection method of the porous membrane material for detecting phosphoinositide prepared by preparation method as claimed in claim 1, is characterized in that, comprises the following steps: 1) put the porous membrane material between the electrochemical cell fixtures, inject electrolyte in the cell, and measure the transmembrane current I ₀ under a predetermined voltage; 2) removing the electrolyte in the electrochemical cell, then adding an actual sample that may contain phosphoinositide, adding the electrolyte with the same content as in step 1) in the actual sample, and measuring the transmembrane current I ₁ under a predetermined voltage; 3) Calculate the value of the current change ratio under the predetermined voltage according to (I ₀ -I ₁ )/I ₀ , if the value of the current change ratio is greater than 0.05, it can be judged that the sample contains phosphoinositide. 4 . The detection method according to claim 3 , characterized in that, before the step of placing the porous membrane material between the electrochemical cell fixtures, it further comprises the step of using an activation solution to balance and activate the porous membrane material. 5 . 5. The detection method of claim 4, wherein the activation solution is pH=2-5 containing 0.05-2.0 mol/L of sodium chloride or potassium chloride solution, wherein the solvent is deionized water. 6 . The detection method according to claim 3 , wherein the pH of the electrolyte in step 1) and step 2) is 2-10. 7 . 7. The detection method of claim 3, wherein the electrode of the electrochemical cell is a silver-silver chloride electrode, a mercury-mercury chloride electrode or a graphite electrode. 8. detection method as claimed in claim 3 is characterized in that, the electrolyte solution of described step 1) and step 2) is to contain the sodium chloride of 0.05-2.0mol/L or potassium chloride solution, and solvent is acetonitrile/ Deionized water solution, the volume ratio of the acetonitrile and deionized water is 0:100-50:50. 9. detection method as claimed in claim 3 is characterized in that, the concrete step of measuring transmembrane current is: adopt picoammeter to collect, power source applies the pulse voltage of -0.2 to +0.2V at both ends of the electrode, each pulse Voltage duration 1-10 seconds.